Parathyroid hormone attenuates low back pain and osteoarthritic pain

ABSTRACT

A method for treating low back pain (LBP) and/or osteoarthritic pain in a subject in need of treatment thereof, the method comprising administering to the subject a composition comprising a recombinant parathyroid hormone (PTH) and a pharmaceutically acceptable carrier is disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/942,945, filed Dec. 3, 2019, the contents of whichare incorporated by reference herein.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 5,918 Byte ASCII (Text) file named“38153-601_ST25,” created on Dec. 2, 2020.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with Government support under AR071432 awardedby the National Institutes of Health (NIH). The Government has certainrights in the invention.

BACKGROUND

Low back pain (LBP) is a common health problem, which most people (80%)experience at some point, especially in older adults. Rubin, 2007;Hartvigsen et al., 2006; Hartvigsen et al., 2004; and Global, regional,and national incidence, prevalence, and years lived with disability for310 diseases and injuries, 1990-2015: a systematic analysis for theGlobal Burden of Disease Study 2015. In the United States alone, thedirect and indirect costs associated with LBP surpass $90 billion peryear, with similar adjusted rates in other countries. Samartzis andGrins, 2017. Ninety percent of LBP is nonspecific LBP, which has noapparent pathoanatomical cause. Krismer and van Tulder, 2007; Koes etal., 2006. Several lumbar structures, such as intervertebral disc, facetjoints, are plausible sources of nonspecific LBP, but the pain cannot bereliably attributed to those structures by clinical tests. Hancock etal., 2007; Maher et al., 2017; and Hartvigsen et al., 2018. Importantly,intervertebral disc (IVD) degeneration is frequently observed inasymptomatic patients, indicating that disc degeneration, per se, is notpainful in some patients. Hurri and Karppinen, 2004; Borenstein et al.,2001. Hence, identifying the source of LBP and related mechanisms isessential to develop effective treatments for LBP.

Further, osteoarthritis (OA) is a leading cause of disability as themost common degenerative joint disorder and chronic pain is the mostprominent symptom of osteoarthritis (OA), affecting nearly 40 millionpeople in the US. Pain itself also is a major risk factor for thedevelopment of future functional limitation and disability in OApatients. Unfortunately, OA pain treatment remains challenging andrepresents a large unmet medical need. It is not clear what causes OApain, and currently there is no effective way to relieve it. Availabletherapies (NSAIDs, steroids, visco-supplementation, such asintra-articular injection of hyaluronic acid) only alleviate mild jointOA pain. Relief from chronic OA pain remains an unmet medical need andstill major reason for seeking surgical intervention. Despite theseefforts, the origins of pain and its molecular mechanisms remain poorlyunderstood.

SUMMARY

In some aspects, the presently disclosed subject matter provides amethod for treating low back pain (LBP) and/or osteoarthritic pain in asubject in need of treatment thereof, the method comprisingadministering to the subject a composition comprising a recombinantparathyroid hormone (PTH) and a pharmaceutically acceptable carrier.

In certain aspects, the low back pain comprises a nonspecific low backpain.

In certain aspects, the administering of the recombinant parathyroidhormone (PTH) inhibits osteoclast activity-induced sensory innervationin a vertebral endplate of the subject.

In other aspects, the administering of the recombinant parathyroidhormone (PTH) increases the intervertebral disc (IVD) space bydecreasing the volume and porosity of sclerotic endplates.

In yet other aspects, the administering of the recombinant parathyroidhormone (PTH) prevents endplate remodeling and sclerosis.

In even yet other aspects, the administering of the recombinantparathyroid hormone (PTH) reduces sensory nerve fibers.

In other aspects, the administering of the recombinant parathyroidhormone (PTH) reduces the porosity of sclerotic endplates.

In particular aspects, the administering of the recombinant parathyroidhormone (PTH) treats the osteoarthritic pain by one or more ofinhibition of nerve innervation, inhibition of subchondral bonedeterioration, inhibition of articular cartilage degeneration,attenuation of joint degeneration, decelerating subchondral bonedeterioration, and sustaining of subchondral bone microarchitecture byremodeling.

In some aspects, the method further comprises administering at least oneother agent in combination with the administering of the recombinantparathyroid hormone (PTH).

In certain aspects, the at least one other agent is selected from thegroup consisting of paracetamol, an opioid, a non-steroidalanti-inflammatory drug, a skeletal muscle relaxant, a triptan, anα2-agonist, a local anesthetic, a tricyclic antidepressant, abenzodiazepine, a steroid, a visco supplement, and combinations thereof.

In particular aspects, the low back pain is associated with one or moreof spine degeneration, lumbar disc herniation (LDH), scoliosis, cancer,and an infection.

In some aspects, the recombinant PTH comprises a full-length PTH proteinor a fragment of PTH. In particular aspects, the recombinant parathyroidhormone comprises teriparatide (PTH(1-34′)). In other aspects, therecombinant parathyroid hormone comprises an intact parathyroid hormone(iPTH).

In certain aspects, the composition is administered to the subject atleast once a day.

In other aspects, the presently disclosed subject matter provides theuse of a recombinant parathyroid hormone to treat low back pain (LBP) orosteoarthritic pain in a subject in need thereof.

Certain aspects of the presently disclosed subject matter having beenstated hereinabove, which are addressed in whole or in part by thepresently disclosed subject matter, other aspects will become evident asthe description proceeds when taken in connection with the accompanyingExamples and Drawings as best described herein below.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

Having thus described the presently disclosed subject matter in generalterms, reference will now be made to the accompanying Figures, which arenot necessarily drawn to scale, and wherein:

FIG. 1-1A, FIG. 1-1B, FIG. 1-1C, FIG. 1-1D, FIG. 1-1E, FIG. 1-1F, FIG.1-1G, FIG. 1-1H, FIG. 1-1I, FIG. 1-1J. FIG. 1-1K, FIG. 1-1L, and FIG.1-1M show symptomatic spinal pain behavior in LSI model and aged mice.(FIG. 1-1A) Pressure hyperalgesia of the lumbar spine was assessed asthe force threshold to induce the vocalization by a force gauge afterLSI or sham surgery. (FIG. 1-1B-FIG. 1-1E) Spontaneous activity analysisincluding distance traveled (FIG. 1-1B), maximum speed (FIG. 1-1C), meanspeed (FIG. 1-1D) and active time (FIG. 1-1E) on the wheel per 24 h,determined by the percentage of sham surgery mice at the correspondingtime points. (FIG. 1-1F, FIG. 1-1G) The hind paw withdrawal frequencyresponding to mechanical stimulation (von Frey, 0.7 mN and 3.9 mN) afterLSI or sham surgery. PWF: Paw Withdraw Frequency. *p<0.05, **p<0.01compared with the sham surgery mice at the corresponding time points.n=6 per group (a-g). (FIG. 1-1H) Pressure hyperalgesia of the low backin 20-month-old or 3-month-old mice. (FIG. 1-1I) The distance traveled.(FIG. 1-1J) mean speed and (FIG. 1-1K) active time on the wheel per 24 hin 20-month-old mice determined by the percentage of 3-month-old mice.(FIG. 1-1L, FIG. 1-1M) The hind paw withdrawal frequency responding tomechanical stimulation (von Frey, 0.7 mN and 3.9 mN) in 20-month-old or3-month-old mice. PWF: Paw Withdraw Frequency. *p<0.05, **p<0.01compared with the 3-month-old mice. n=8 per group (h-m). Statisticalsignificance was determined by multifactorial ANOVA, and all data areshown as means±standard deviations;

FIG. 1-2A, FIG. 1-2B, FIG. 1-2C, FIG. 1-2D, FIG. 1-2E, and FIG. 1-2Fdemonstrate that sensory innervation in endplates correlates withincrease of osteoclasts in LSI model. (FIG. 1-2A) Representative imagesof coronal mouse caudal endplate sections of L4/5 stained for TRAP(magenta) at 2, 4 and 8 weeks after LSI or sham surgery. Scale bars, 50μm. (FIG. 1-2B) Quantitative analysis of the number of TRAP⁺ cells inendplates. (FIG. 1-2C) Representative immunofluorescent images of CGRP⁺sensory nerve fibers (red) and DAPI (blue) staining of nuclei in mousecaudal endplates of L4/5 at 2, 4 and 8 weeks after LSI or sham surgery.Scale bars, 50 μm. (FIG. 1-2D) Percentage of CGRP⁺ area in endplates.(FIG. 1-2E) Representative images of immunofluorescent analysis of CGRP⁺(red), PGP9.5⁺ (green) nerve fibers and DAN (blue) staining of nuclei inmouse caudal endplates of L4/5 at 8 weeks after LSI or sham surgery.Scale bars, 50 μm. (FIG. 1-2F) Representative images ofimmunofluorescent analysis of IB4⁺ (green) sensory nerve fibers and DAPI(blue) staining of nuclei in mouse caudal endplates of L4/5 at 2, 4 and8 weeks after LSI or sham surgery. Scale bars, 100 μm. **p<0.01 comparedwith the sham surgery mice at the corresponding time points. n=6 pergroup (FIG. 1-2B, FIG. 1-2D). Statistical significance was determined bymultifactorial ANOVA, and all data are shown as means±standarddeviations;

FIG. 1-3A, FIG. 1-3B, FIG. 1-3C, FIG. 1-3D, FIG. 1-3E, FIG. 1-3F, FIG.1-3G, FIG. 1-3H, and FIG. 1-31 demonstrate that sensory innervation inendplates correlates with increase of osteoclasts during aging. (FIG.1-3A) Representative three-dimensional high-resolution μCT images of themouse caudal endplates of L4/5 (coronal view) in 3-month-old and20-month-old mice. Scale bars, 1 mm. (FIG. 1-3B-FIG. 1-3C) Quantitativeanalysis of the total porosity (FIG. 1-3B) and trabecular separation(Tb. Sp; FIG. 1-3C) of the mouse caudal endplates of L4/5 determined byμCT. (FIG. 1-3D) Top and middle, representative images of safranin O andfast green staining of coronal caudal endplate sections of L4/5 in3-month-old and 20-month-old mice, proteoglycan (red) and cavities(green). Bottom, representative images of TRAP (magenta) staining ofcoronal sections of the caudal endplates of L4/5 in 3-month-old and20-month-old mice. Scale bars, 50 μm (FIG. 1-3E) Endplate scores in3-month-old and 20-month-old mice as an indication of endplatedegeneration based on safranin O and fast green staining. (FIG. 1-3F)Quantitative analysis of the number of TRAP⁺ cells in endplates. (FIG.1-3G) Representative images of immunofluorescent analysis of CGRP⁺sensory nerve fibers (red) and DAPI (blue) staining of nuclei in mousecaudal endplates of L4/5 in 20-month-old and 3-month-old mice. Scalebars, 50 μm. (FIG. 1-3H) Quantitative analysis of the percentage ofCGRP⁺ area in endplates. (FIG. 1-3I) Representative images ofimmunofluorescent analysis of CGRP⁺ (green), PGP9.5⁺ (red) nerve fibersand DAPI (blue) staining of nuclei in mouse caudal endplates of L4/5 in20-month-old mice. Scale bars, 50 μm. *p<0.05. **p<0.01 compared withthe 3-month-old mice. n=6 per group (FIG. 1-3B, FIG. 1-3C, FIG. 1-3E,FIG. 1-3F, and FIG. 1-3H). Statistical significance was determined bytwo-tailed Student's t-test, and all data are shown as means±standarddeviations:

FIG. 1-4A, FIG. 1-4B, FIG. 1-4C, FIG. 1-4D, FIG. 1-4E, FIG. 1-4F, FIG.1-4G, FIG. 1-4H, and FIG. 1-4I demonstrate that sensory innervation inendplates is validated by retrograde and anterograde tracing. (FIG.1-4A) Model of retrograde tracing of the sensory innervation in theendplates of L4/5. The T12-L6 DRGs were harvested at 1 week afterinjection of Dil in the left part of the mouse caudal endplates at 8weeks after LSI or sham surgery. (FIG. 1-4B) Representative images ofDil⁺ (red) sensory neurons and DAPI (blue) staining of nuclei in theleft (L) and right (R) side DRGs. Scale bars, 200 μm. (FIG. 1-4C)Quantitative analysis of the number of Dil cells of (b). **p<0.01compared with the sham surgery mice at the corresponding side. n=6 pergroup. (FIG. 1-4D) Top, representative images of Dil⁺ (red) and CGRP⁺sensory neurons and DAPI (blue) staining of nuclei in the left (L) sideDRGs of L1 and L2. Bottom, representative images of Dil⁺ (red) and IB4⁺sensory neurons and DAPI (blue) staining of nuclei in the left (L) sideDRGs of L1 and L2. Scale bars, 100 μm. (FIG. 1-4E) Quantitative analysisof (d). **p<0.01 compared with the percentage of Dil^(+IB)4⁺ cells toDil⁺ cells in the corresponding DRG. n=6 per group. (FIG. 1-4F-FIG.1-4H) The anterograde tracing analysis of L1 and L2 DRG neuronal fibersinnervation into the caudal endplates of L4/5. The Dil was injected inthe L1 and L2 DRGs at 8 weeks after LSI and sham surgery; or in3-month-old and 20-month-old mice. Representative images of Dil⁺ (red)sensory nerve fibers in the caudal endplates of L4/5 in LSI and shamsurgery mice (FIG. 1-4F) or in 3-month-old and 20-month-old group (FIG.1-4H) at 1 week after injection. Scale bars, 100 μm. Quantitativeanalysis of the percentage of Dil⁺ area in endplates in LSI and shamsurgery group (FIG. 1-4G) or in 3-month-old and 20-month-old group (FIG.1-4I). **p<0.01 compared with the 3-month-old mice. n=6 per group (FIG.1-4G and FIG. 1-4I). Statistical significance was determined bymultifactorial ANOVA, and all data are shown as means f standarddeviations;

FIG. 1-5A, FIG. 1-5B, FIG. 1-5C, FIG. 1-5D, FIG. 1-5E, FIG. 1-5F, FIG.1-5G, FIG. 1-5H, and FIG. 1-5I demonstrate that PGE2/EP4 contributes tothe spinal pain hypersensitivity. (FIG. 1-54 ) Quantitative analysis ofthe expression of PGE synthetase (PGES), cox2, IL-1β, IL-17, IL-2 andTNF-α in lumbar endplates at 4 weeks after LSI determined byqRT-PCR_(FIG. 1-5B) Representative images of Immunohistochemicalanalysis of Cox2 (brown; top) or PGE2 (brown; bottom) in the caudalendplates of L4/5 at 4 and 8 weeks after LSI or sham surgery. Scalebars, 50 μm. (FIG. 1-5C) ELISA analysis of PGE2 concentration in thelysate of lumbar endplates at 4.8 and 12 weeks after LSI surgery.*p<0.05, **p<0.01 compared with the sham surgery mice. n=3 (FIG. 1-5Aand FIG. 1-5C). (FIG. 1-5D) Representative images of immunofluorescentanalysis of CGRP (red), EP4 (green) staining and DAPI (blue) staining ofnuclei in the caudal endplates of L4/5 at 4 and 8 weeks after LSIsurgery. Scale bars, 50 μm. (FIG. 1-5E) Representative images ofimmunofluorescent analysis of CGRP (red), EP4 (green) staining and DAPI(blue) staining of nuclei in the L2 DRGs at 4 and 8 weeks after LSIsurgery. Scale bars, 100 μm. (FIG. 1-5F) Quantitative analysis ofpercentage of CGRP⁺ EP4⁺ cells to CGRP cells in the L2 DRGs at 4 and 8weeks after LSI surgery. (FIG. 1-5G) Representative images ofimmunofluorescent analysis of CGRP (red), Na_(v) 1.8 (green) stainingand DAPI (blue) staining of nuclei in the caudal endplates of L4/5 at 4weeks after LSI surgery. Scale bars, 50 μm. (FIG. 1-5H) Representativeimages of immunofluorescent analysis of CGRP (red). Na_(v) 1.8 (green)staining and DAPI (blue) staining of nuclei in the L2 DRGs at 4 and 8weeks after LSI surgery. Scale bars, 100 μm. (FIG. 1-5I) Quantitativeanalysis of percentage of CGRP⁺ Na_(v) 1.8⁺ cells to CGRP⁺ cells in theL2 DRGs at 4 and 8 weeks after LSI surgery. **p<0.01 compared with thesham surgery mice at the corresponding time points. n=6 per group (FIG.1-5F and FIG. 1-5I). Statistical significance was determined bymultifactorial ANOVA, and all data are shown as means f standarddeviations;

FIG. 1-6A, FIG. 1-6B, FIG. 1-6C, FIG. 1-6D, FIG. 1-6E, FIG. 1-6F, FIG.1-6G, FIG. 1-6H, FIG. 1-6I, FIG. 1-6J, and FIG. 1-6K demonstrates thatPGE2 stimulates PKA/CREB signaling through EP4 to induce sodium influx.(FIG. 1-6A) Representative images of sodium indicator (green) analysispre- and post-PGE2 (20 μM) stimulation for 5 min in primary DRG neuronsfrom EP4^(f/f) for EP4^(−/−) mice, indicating sodium influx. Scale bar,100 μm. Magnification, scale bar, 20 μm. (FIG. 1-6B, FIG. 1-6C)Quantitative analysis of the fluorescent density distribution of the1^(st) (FIG. 1-6B) and 2^(nd) (FIG. 1-6C) column in (FIG. 1-6A).*p<0.05, **p<0.01 compared with the corresponding pre-treatment group.n=3 per group. (FIG. 1-6D) Western blots of the phosphorylation of PKAand CREB in primary DRG neurons treated with PGE2 (20 LM) for 30 min andPKA inhibitor (H-89, 10 μM) for 60 min. (FIG. 1-6E) Quantitativeanalysis of (FIG. 1-6D). **p<0.01 compared with the negative controlgroup from EP4^(f/f) mice. ^(#)p<0.05, ^(#)p<0.01 compared with onlyPGE2 treatment group from EP4^(f/f) mice n=3 per group. (FIG. 1-6F)1^(st) to 3^(rd) row, representative images of immunofluorescentanalysis of PKA (red), p-PKA (green) staining and DAPI (blue) stainingof nuclei: 4^(th) to 6^(th) row, representative images ofimmunofluorescent analysis of CREB (red), p-CREB(green) staining andDAPI (blue) staining of nuclei pre- and post-PGE2 (20 μM) stimulationcombined with H-89 (10 μM) in primary DRG neurons from EP4^(f/f) orEP4^(−/−) mice. Scale bar, 100 μm. (FIG. 1-6G) Representative images ofsodium indicator (green) analysis pre- and post-PGE2 (20 μM) stimulationcombined with cAMP, PKA inhibitor (H-89) or siRNA for Na_(v) 1.8(si-Na_(v)1.8) in primary DRG neurons from EP4^(f/f) for EP4^(−/−) mice.Scale bar, 100 μm. Magnification, scale bar, 20 μm. (FIG. 1-6H-FIG.1-6K) Quantitative analysis of the fluorescent density distribution ofthe 1^(st) (FIG. 1-6H), 2^(nd) (FIG. 1-6I), 3^(rd) (FIG. 1-6J), 4^(th)(FIG. 1-6K) column in (FIG. 1-6G). *p<0.05, **p<0.01 compared with thecorresponding pre-treatment group. n=3 per group. Statisticalsignificance was determined by multifactorial ANOVA, and all data areshown as means t standard deviations;

FIG. 1-7A. FIG. 1-7B. FIG. 1-7C, FIG. 1-7D, FIG. 1-7E, FIG. 1-7F, andFIG. 1-7G demonstrate that EP4 knockout in sensory nerve attenuatesspinal pain behavior. (FIG. 1-7A-FIG. 1-7G) Quantitative analysis ofspinal pain-related behavior tests, including pressure hyperalgesia(FIG. 1-7A), spontaneous distance travelled (FIG. 1-7B), maximum speed(FIG. 1-7C), mean speed (FIG. 1-7D), active time (FIG. 1-7E) per 24hours and hind paw withdrawal frequency responding to mechanicalstimulation (0.7 mN: FIG. 1-7F and 3.9 mN; FIG. 1-7G) in EP4 orEP4^(f/f) mice overtime after LSI or sham surgery. PWF: Paw withdrawfrequency. *p<0.05. **p<0.01 compared with EP4^(f/f) sham surgery mice,^(#)p<0.05, ^(##)p<0.01 compared with EP4^(f/f) LSI surgery mice at thecorresponding time points. n=8 per group (FIG. 1-7A-FIG. 1-7G).Statistical significance was determined by multifactorial ANOVA, and alldata are shown as mean±standard deviations;

FIG. 1-8A, FIG. 1-8B, FIG. 1-8C, FIG. 1-8D, FIG. 1-8E, FIG. 1-8F, FIG.1-8G, FIG. 1-8H. FIG. 1-81 , FIG. 1-8J, FIG. 1-8K. FIG. 1-8L, FIG. 1-8M,FIG. 1-8N, FIG. 1-8O, and FIG. 1-8P show that decreased osteoclastsactivity diminishes sensory innervation and attenuates pain. (FIG. 1-8A)Representative μCT images of the caudal endplates of L4/5 (coronal view)in Rankl^(−/−) or Rankl^(f/f) mice at 4 and 8 weeks after LSI or shamsurgery. Scale bars, 1 mm. (FIG. 1-8B-FIG. 1-8C) Quantitative analysisof the total porosity (FIG. 1-8B) and trabecular separation (Th. Sp;FIG. 1-8C) of the mouse caudal endplates of L4/5 determined by μCT.(FIG. 1-8D) Representative images of safranin O and fast green stainingof coronal sections of the caudal endplates of L4/5 in Rankl^(−/−) orRankl^(f/f) mice at 4 and 8 weeks after LSI or sham surgery. Scale bars,50 μm. (FIG. 1-8E) Endplate scores of the caudal endplates. (FIG. 1-8F)Representative images of TRAP (magenta) staining of coronal sections ofthe caudal endplates of L4/5 in Rankl^(−/−) or Rankl^(f/f) mice at 4 and8 weeks after LSI or sham surgery. Scale bars, 50 μm. (FIG. 1-8G)Quantitative analysis of the number of TRAP⁺ cells in the caudalendplates. (FIG. 1-8H) Representative images of immunofluorescentanalysis of CGRP⁺ sensory nerve fibers (red) and DAPI (blue) staining ofnuclei in caudal endplates of L4/5 in Rankl^(−/−) or Rankl^(f/f) mice at4 and 8 weeks after LSI or sham surgery. Scale bars, 50 μm. (FIG. 1-8I)Quantitative analysis of the percentage of CGRP⁺ area in caudalendplates. (FIG. 1-8J-FIG. 1-8P) Quantitative analysis of spinepain-related behavior tests, including pressure hyperalgesia (FIG.1-8J), distance travelled (FIG. 1-8K), maximum speed (FIG. 1-8L), meanspeed (FIG. 1-8M), active time (FIG. 1-8N) per 24 hours and hind pawwithdrawal frequency responding to mechanical stimulation (0.7 mN; FIG.1-8O and 3.9 mN; FIG. 1-8P) in Rankl^(−/−) or Rankl^(f/f) mice overtimeafter LSI or sham surgery. **p<0.01 compared with Rankl^(f/f) shamsurgery mice, **p<0.01 compared with Rankl^(f/f) LSI surgery mice at thecorresponding time points. n=8 per group (FIG. 1-8B. FIG. 1-8C, FIG.1-8E, FIG. 1-8G, and FIG. 1-81 -FIG. 1-8P). Statistical significance wasdetermined by multifactorial ANOVA, and all data are shown asmeans±standard deviations;

FIG. 1-9A, FIG. 1-9B, FIG. 1-9C, FIG. 1-9D, FIG. 1-9E, FIG. 1-9F, FIG.1-9G, FIG. 1-9H, FIG. 1-91 , FIG. 1-9J, FIG. 1-9K, FIG. 1-9L, FIG. 1-9M,FIG. 1-9N, FIG. 1-9O, and FIG. 1-9P demonstrate that knockout ofnetrin-1 abrogates sensory innervation and spinal pain. (FIG. 1-9A)Representative images of immunofluorescent analysis of TRAP⁺ (red),Netrin-1⁺ (green) and DAPI (blue) staining of nuclei in caudal endplatesof L4/5 after LSI or sham surgery. Scale bars, 50 m. (FIG. 1-9B) ELISAanalysis of Netrin-1 concentration in the lysate of lumbar endplatesafter LSI surgery. **p<0.01 compared with the sham surgery mice. n=3 pergroup. (FIG. 1-9C) Representative images of immunofluorescent analysisof CGRP⁺ (red), DCC⁺ (green) and DAPI (blue) staining of nuclei incaudal endplates of L4/5 after LSI surgery. Scale bars, 50 μm. (FIG.1-9D) Representative images of safranin O and fast green staining of thecaudal endplates of L4/5 in Netrin-1^(−/−) or Netrin-1^(f/f) mice afterLSI or sham surgery. Scale bars, 50 μm. (FIG. 1-9E) Endplate scores ofthe caudal endplates. (FIG. 1-9F) Representative images of TRAP(magenta) staining of the caudal endplates of L4/5 in Netrin-1^(−/−) orNetrin-1^(f/f) mice after LSI or sham surgery. Scale bars, 50 μm. (FIG.1-9G) Quantitative analysis of (FIG. 1-9F). (FIG. 1-9H) Representativeimages of immunofluorescent analysis of CGRP⁺ (red) and DAPI (blue)staining of nuclei in caudal endplates of L4/5 in Netrin-1^(−/−) orNetrin-1^(f/f) mice after LSI or sham surgery. Scale bars, 50 μm. (FIG.1-9I) Quantitative analysis of (FIG. 1-9H). (FIG. 1-9J-FIG. 1-9P)Quantitative analysis of spinal pain-related behavior tests, includingpressure hypersensitivity (FIG. 1-9J), distance travelled (FIG. 1-9K),maximum speed (FIG. 1-9L), mean speed (FIG. 1-9M), active time (FIG.1-9N) per 24 hours and hind paw withdrawal frequency responding tomechanical stimulation (0.7 mN; FIG. 1-9O and 3.9 mN; FIG. 1-9P) inNetrin-1^(−/−) or Netrin-1^(f/f) mice after LSI or sham surgery.*p<0.05. **p<0.01 compared with Netrin-If sham surgery mice, ^(#)p<0.05,^(#)p<0.01 compared with Netrin-1^(f/f) LSI surgery mice at thecorresponding time points, ns, no significant difference, compared withNetrin-If LSI surgery mice at the corresponding time points. n=7 pergroup (FIG. 1-9G, FIG. 1-91 -FIG. 1-9P). Statistical significance wasdetermined by multifactorial ANOVA, and all data are shown as means fstandard deviations:

FIG. 1-10A and FIG. 1-10B show the formation of CD31⁺Emcn⁺ vessels inEndplate during spinal instability. (FIG. 1-10A) Representative imagesof immunofluorescent analysis of CD31⁺ (green). Emcn⁺ (red) and DAPI(blue) staining of nuclei in mouse caudal endplates of L4/5 at 2, 4 and8 weeks after LSI or sham surgery. Scale bars, 50 μm. (FIG. 1-10B)Percentage of CD31⁺Emcn⁺ area in endplates. **p<0.01 compared with thesham surgery mice at the corresponding time points. n=6 per group.Statistical significance was determined by multifactorial ANOVA, and alldata are shown as means±standard deviations;

FIG. 1-11A, FIG. 1-11B, FIG. 1-11C, FIG. 1-11D, and FIG. 1-11E show theμCT analysis of the vertebral trabecular bone during aging. (FIG. 1-11A)Representative three-dimensional high-resolution μCT images of thetrabecular bone of L5 vertebrae (coronal view) in 3-month-old and20-month-old mice. Scale bars, 1 mm. (FIG. 1-11B-FIG. 1-11E)Quantitative analysis of the trabecular bone volume/total volume (BV/TV)(FIG. 1-11B) and trabecular bone number (Tb.N, FIG. 1-11C), trabecularbone thickness (Tb.Th, FIG. 1-11D), and trabecular bone separationdistribution (Th. Sp, FIG. 1-11E) in L5 vertebrae determined by μCT.**p<0.01, ns, not significant difference compared with the 3-month-oldmice. n=6 per group. Statistical significance was determined bytwo-tailed Student's t test, and all data are shown as mean±standarddeviations:

FIG. 1-12A and FIG. 1-12B show the formation of CD31⁺Emcn⁺ vessels inEndplate during aging. (FIG. 1-12A) Representative images ofimmunofluorescent analysis of CD31⁺ (green), Emcn⁺ (red) and DAPI (blue)staining of nuclei in mouse caudal endplates of L4/5 in 20-month-old and3-month-old mice. Scale bars, 50 μm. (FIG. 1-12B) Quantitative analysisof the percentage of CD31⁺Emcn⁺ area in endplates. **p<0.01 comparedwith the 3-month-old mice. n=6 per group. Statistical significance wasdetermined by two-tailed Student's t test, and all data are shown asmeans±standard deviations;

FIG. 1-13A, FIG. 1-13B, FIG. 1-13C, and FIG. 1-13D demonstrate nerveinnervation in the human sclerotic endplates. (FIG. 1-13A)Representative gross appearance (top) and images of safranin O and fastgreen staining of coronal sections (bottom) of the endplates frompatients without LBP or with LBP. Scale bars, 50 μm. (FIG. 1-13B)Endplate scores of the samples from patients without LBP or with LBP.(FIG. 1-13C) Representative images of TRAP (magenta) staining of coronalsections of the endplates from patients without LBP or with LBP. Scalebars, 50 μm. (FIG. 1-13D) Representative immunofluorescent images ofCGRP⁺ (red), PGP9.5⁺ (green) and DAPI (blue) staining of nuclei in theendplates. Scale bars, 50 μm. **p<0.01 compared with patients withoutLBP. n=6 of non-LBP group, n=9 of LBP group. LBP: low back pain.Statistical significance was determined by two-tailed Student's t test,and all data are shown as means±standard deviations;

FIG. 1-14 shows the potential source of PGE2 in porous endplates.Representative immunofluorescent images of cox2⁺ (red) and F4/80⁺(green). cox2⁺ (red) and OCN⁺ (green), cox2⁺ (red) and TRAP⁺ (green) andDAPI (blue) staining of nuclei in the endplates. Scale bars, 50 μm;

FIG. 1-15A and FIG. 1-15B show that EP4 knockout did not affect theLSI-induced increase in the number of TRAP⁺ osteoclasts in endplates.(FIG. 1-15A) Representative images of coronal mouse caudal endplatesections of L4/5 stained for TRAP (magenta) at 8 weeks after LSI or shamsurgery in EP4^(f/f) and EP4^(−/−) mice. Scale bars, 50 μm. (FIG. 1-15B)Quantitative analysis of the number of TRAP⁺ cells in endplates.**p<0.01 compared with sham surgery group. ns, not significantdifference compared with sham surgery mice in corresponding transgenicgroup. n=6 per group. Statistical significance was determined bymultifactorial ANOVA, and all data are shown as means±standarddeviations;

FIG. 1-16A, FIG. 1-16B, FIG. 1-16C, FIG. 1-16D, and FIG. 1-16E show theosteopetrotic phenotype of Vertebrae in Rankl^(−/−) mice. (FIG. 1-16A)Representative μCT images of the trabecular bone (coronal view) in L5vertebrae of Rankl^(−/−) and Rankl^(f/f) mice in sham or LSI surgerygroup. Scale bars, 1 mm. (FIG. 1-16B-FIG. 1-16E) Quantitative analysisof the Trabecular BV/TV (FIG. 1-16B), Tb.N (FIG. 1-16C), Tb.Th (FIG.1-16D), and Tb. Sp (FIG. 1-16E) of the mouse L5 vertebrae determined byμCT. BV, Bone Volume. TV, Total Volume. Tb.N, Trabecular bone Number.Tb.Th, Trabecular bone Thickness. Tb. Sp, Trabecular Separationdistribution. **p<0.01 compared with Rankl^(f/f) mice. ns, nosignificant difference compared with sham surgery mice in correspondingtransgenic group. n=6 per group. Statistical significance was determinedby multifactorial ANOVA, and all data are shown as means±standarddeviations;

FIG. 1-17A and FIG. 1-17B demonstrate that decreased osteoclastsactivity diminished the formation of CD31⁺Emcn⁺ vessels in Endplate.(FIG. 1-17A) Representative images of immunofluorescent analysis ofCD31⁺ (green), Emcn⁺ (red) and DAPI (blue) staining of nuclei in caudalendplates of L4/5 in Rankl^(−/−) or Rankl^(f/f) mice at 4 and 8 weeksafter LSI or sham surgery. Scale bars, 50 μm. (FIG. 1-17B) Quantitativeanalysis of the percentage of CD31⁺Emcn⁺ area in caudal endplates.**p<(0.01 compared with Rankl^(f/f) sham surgery mice, ^(##)p<0.01compared with Rankl^(f/f) LSI surgery mice at the corresponding timepoints n=8 per group. Statistical significance was determined bymultifactorial ANOVA, and all data are shown as means f standarddeviations;

FIG. 1-18A and FIG. 1-18B demonstrate that reduction of osteoclastactivity did not inhibit sensory innervation in the annulus fibrosus inLSI mice. (FIG. 1-18A) Representative immunofluorescent images of CGRP⁺sensory nerve fibers (red) and DAPI (blue) staining of nuclei in mouseannulus fibrosus of L4/5 at 8 weeks after LSI or sham surgery. Scalebars, 100 μm. (FIG. 1-18B) Percentage of CGRP⁺ area in annulus fibrosus.**p<0.01 compared with sham surgery mice, ns, not significantdifference, compared with Rankl^(f/f) LSI surgery mice. n=6 per group.Statistical significance was determined by multifactorial ANOVA, and alldata are shown as means±standard deviations;

FIG. 1-19A and FIG. 1-19B demonstrate that knockout of netrin-1 in theTRAP+ lineage cells inhibited the formation of CD31⁺Emcn⁺ vessels inEndplate. (FIG. 1-19A) Representative images of immunofluorescentanalysis of CD31⁺ (green), Emcn⁺ (red) and DAPI (blue) staining ofnuclei in caudal endplates of L4/5 in Netrin-1^(−/−) or Netrin-1^(f/f)mice at 4 and 8 weeks after LSI or sham surgery. Scale bars, 50 μm.(FIG. 1-19B) Quantitative analysis of the percentage of CD31⁺Emcn⁺ areain caudal endplate. **p<0.01 compared with Netrin-1^(f/f) sham surgerymice, ^(##)p<0.01 compared with Netrin-1^(f/f) LSI surgery mice at thecorresponding time points. n=7 per group. Statistical significance wasdetermined by multifactorial ANOVA, and all data are shown asmeans±standard deviations;

FIG. 1-20A and FIG. 1-20B demonstrate that knockout of Netrin-1 in theTrap⁺ cells did not inhibit sensory innervation in the annulus fibrosusin LSI mice. (FIG. 1-20A) Representative immunofluorescent images ofCGRP⁺ sensory nerve fibers (red) and DAPI (blue) staining of nuclei inmouse annulus fibrosus of L4/5 at 8 weeks after LSI or sham surgery.Scale bars, 100 μm. (FIG. 1-20B) Percentage of CGRP⁺ area in annulusfibrosus. **p<0.01 compared with sham surgery mice, ns, not significantdifference, compared with Netrin-1^(f/f) LSI surgery mice. n=6 pergroup. Statistical significance was determined by multifactorial ANOVA,and all data are shown as means±standard deviations:

FIG. 2-1A, FIG. 2-1B, FIG. 2-1C, FIG. 2-1D, FIG. 2-1E, FIG. 2-1F, FIG.2-1G, FIG. 2-1H, FIG. 2-1I, and FIG. 2-1J demonstrate that iPTHattenuated low back pain related behavior test in LSI mouse model andAging Mice. (FIG. 2-1A) Lumbar spine instability mouse model (LSI) andAging mouse (FIG. 2-1F) had been done the surgery or treatment accordingto the schedule (FIG. 2-1B, FIG. 2-1G) Pressure hyperalgesia of thelumbar spine assessed as the force threshold to induce the vocalizationby a force gauge in LSI mouse model (FIG. 2-1B) and Aging model (FIG.2-1G). (FIG. 2-1C-FIG. 2-1E, FIG. 2-1H-FIG. 2-1J) Spontaneous activityanalysis including active time (FIG. 2-1C, FIG. 2-1H), distance traveled(FIG. 2-1D, FIG. 2-1I) and mean speed (FIG. 2-1E, FIG. 2-1J) on thewheel per 24 h in Sham, iPTH or vehicle treatment group. *p<0.05,**p<0.01 compared with the sham surgery mice or vehicle group at thecorresponding time points. n=8 per group. Statistical significance wasdetermined by multifactorial ANOVA or T-test, and all data are shown asmeans±standard deviation;

FIG. 2-1A, FIG. 2-1B, FIG. 2-1C, FIG. 2-1D, FIG. 2 -IE, FIG. 2-1F, FIG.2-1G, FIG. 2-1H, FIG. 2-1I, and FIG. 2-1J show that iPTH attenuated lowback pain related behavior test in LSI mouse model and Aging Mice (FIG.2-1A) Lumbar spine instability mouse model (LSI) and (FIG. 2-1F) Agingmouse had been done the surgery or treatment according to the schedule.(FIG. 2-1B-FIG. 2-1J) Spontaneous activity analysis including distancetraveled (FIG. 2-1B, FIG. 2-1G), mean speed (FIG. 2-1C, FIG. 2-1H) andactive time (FIG. 2-1D, FIG. 2-1I) on the wheel per 24 h in Sham, iPTHor vehicle treatment group. (FIG. 2-1E, FIG. 2-1J) Pressure hyperalgesiaof the lumbar spine assessed as the force threshold to induce thevocalization by a force gauge in LSI mouse model (FIG. 2-1E) and Agingmodel (FIG. 2-1J). *p<0.05, **p<0.01 compared with the sham surgery miceor vehicle group at the corresponding time points. n=8 per group.Statistical significance was determined by multifactorial ANOVA orT-test, and all data are shown as means±standard deviations;

FIG. 2-2A, FIG. 2-2B, FIG. 2-2C, FIG. 2-2D, FIG. 2-2E, FIG. 2-2F, FIG.2-2G, FIG. 2-2H demonstrate that PTH increased the IVD space bydecreasing the volume and porosity of sclerotic endplates. (FIG. 2-2A)(Top) Representative coronal high-resolution microcomputed tomography(μCT) images and (Bottom) three-dimensional images of the L4/5 mouseendplates in Sham surgery group, LSI mice treated with vehicle or iPTHgroups, and Aging mice treated with vehicle or iPTH groups. Scale bars,0.5 mm. (FIG. 2-2B) Representative images of safranin O and fast greenstaining of coronal endplate sections of L4/5 in Sham, LSI treated withvehicle or PTH, Aging treated with vehicle or PTH groups, proteoglycan(red) and cavities (green). Scale bars, 100 m. (FIG. 2-2C-FIG. 2-2E)Quantitative analysis of the total porosity (FIG. 2-2C), endplate volume(FIG. 2-2D) and volume of porosity (FIG. 2-2E) of the mouse L4/5endplate. (FIG. 2-2F) Quantitative analysis of the area of cartilage inthe endplate based on safranin O and fast green staining as anindication of cartilage endplate degeneration. (FIG. 2-2G-FIG. 2-2H)Quantification of IVD height and endplate thickness in the back 1/3 ofL4/L5 sagittal plane. n=8 per group. Data are shown as mean±s.d.*p<0.05. **p<0.01;

FIG. 2-3A, FIG. 2-3B, FIG. 2-3C, FIG. 2-3D, FIG. 2-3E, FIG. 2-3F, FIG.2-3G, FIG. 2-3H demonstrate that sensory innervation decreased in PTHremodeling of sclerotic endplates. (FIG. 2-3A-FIG. 2-3B) Representativeimages of immunofluorescent analysis of PGP9.5+(green) nerve fibers andDAPI (blue) staining of nucleus in mouse endplates (B 1.2) and annularfibrosis (B 3) of L4/5 of Sham groups, LSI treated with Vehicle or PTHgroups, and Aging treated with Vehicle or PTH groups Scale bars, 50 μm.(FIG. 2-3C-FIG. 2-3D) Quantitative analysis of the percentage of PGP9.5+area in endplates (FIG. 2-3C) or annular fibrosis (FIG. 2-3D). (FIG.2-3E-FIG. 2-3G) Retrograde tracing of the sensory innervation in theendplates of L4/5 with DIL and quantitative analysis of L1-2 DRGimmuno-stained different sensory nerve fibers. Representative images ofDil+(red) sensory neuron of L1-2 DRG sections, DAPI (blue) staining ofnuclei and different sensory nerve fiber markers (green) includingPGP9.5, CGRP, IB4, P2X3, PIEZO2 or NF200 respectively (FIG. 2-3E), Scalebars, 100 μm. Quantitative analysis of the number of Dil+ cells ofPGP9.5+ Cells (FIG. 2-3F) in Sham groups, LSI treated with Vehicle orPTH groups, and Aging treated with Vehicle or PH groups. Quantitativeanalysis of the percentage of Dil+NF200+. Dil+PIZO2+ or Dil+CGRP+ cellsto Dil+ cells in the corresponding DRG. n=5 per group (FIG. 2-3G).Statistical significance was determined by multifactorial ANOVA, and alldata are shown as means±standard deviations. *p<0.05, **p<0.01;

FIG. 2-4A, FIG. 2-4B, FIG. 2-4C, FIG. 2-4D, FIG. 2-4E, FIG. 2-4F, FIG.2-4G, FIG. 2-4H, and FIG. 2-4I demonstrate that bone remodeling reducesthe porosity of sclerotic endplate after iPTH (FIG. 2-4A-FIG. 2-4C)Representative images of TRAP (Red) staining (FIG. 2-4A),immunohistology of Osteocalcin (FIG. 2-4B), immunofluorescent analysisof CD31+ (green), Emcn+ (red) and DAPI (blue) staining (FIG. 2-4C) ofcoronal sections of the endplates of L4/5 in Sham, LSI treated withVehicle or PTH groups, Aging treated with Vehicle or PTH groups. Scalebars, 50 m. (FIG. 2-4D-FIG. 2-4E) Representative images of doublelabeling with Calcein/Alizarin red (FIG. 2-4D), and Goldner staining(FIG. 2-4E) in the L4/5 endplate of Sham, LSI treated with Vehicle orPTH groups, Aging treated with Vehicle or PH groups. Scale bars, 50 m.(FIG. 2-4F-FIG. 2-4H) Quantitative analysis of the number of TRAP+ cells(FIG. 2-4F), the number of osteocalcin+ cells (FIG. 2-4G), thepercentage of CD31+Emcn+ area in endplates (FIG. 2-4H). (FIG. 2-4I) Axonattractive or repulsive factors including netrin-1, sema3a, slit1, sli2,slit3, NGF, and the inflammatory factors including COX-2,PGES, IL-1,TNF-α expression in lumbar endplates at LSI with vehicle or PTHtreatment determined by qRT-PCR. Data are shown as mean±s.d. *p<0.05.**p<0.01;

FIG. 2-5A, FIG. 2-5B, FIG. 2-5C, FIG. 2-5D, FIG. 2-5E, and FIG. 2-5Fdemonstrates that lower porosity endplate caused by iPTH was better tosupport the mechanical stress, which resulted in lower expression ofCOX-2 and PGE2. (FIG. 2-5A-FIG. 2-5C) Representative images of Finiteelement analysis including Miser stress and U magnitude (FIG. 2-5A), andimmunohistochemical analysis of COX-2 (brown; Top) or PGE2 (brown:Bottom) in L4/5 upper endplates of sham group. LSI treated with Vehicleor PTH groups, and Aging treated with Vehicle or PTH groups. Scale bars,50 μm. (FIG. 2-5C-FIG. 2-5F) Quantitative analysis of the Mister stress(FIG. 2-5C), the percentage of COX-2+ cells (FIG. 2-5D) or PGE2+ cells(FIG. 2-5E) in mouse L4/5 endplates. (FIG. 2-5F) ELISA analysis of PGE2concentration in lysate of lumbar endplates. *p<0.05, **p<0.01 n=3 pergroup, statistical significance was determined by multifactorial ANOVA,and all data are shown as mean±s.d:

FIG. 2-6A, FIG. 2-6B, FIG. 2-6C, FIG. 2-6D, FIG. 2-6E, FIG. 2-6F, FIG.2-6G, FIG. 2-6H, FIG. 2-61 , FIG. 2-6J, FIG. 2-6K, and FIG. 2-6Ldemonstrate that iPTH attenuates endplate sclerosis and discdegeneration in aging monkey. (FIG. 2-6A-FIG. 2-6B) Representative T2weighted MR images of same monkey in each group with vehicle (Top) orPTH (Bottom) treated 0 months, 3 months, and 6 months (FIG. 2-6A).Quantitative analysis of the change of Pfirrmann grade of same segmentat same monkey with vehicle or PTH treated 0 months, 3 months, and 6months (FIG. 2-6B). (FIG. 2-6C-FIG. 2-6E) Representative images ofmeasuring T1ρ or T2 map value in nucleus pulposus (FIG. 2-6C).Quantitative analysis of T1ρ value (FIG. 2-6D) or T2 map value (FIG.2-6E) in nucleus pulposus of the same monkey in each group. (FIG.2-6F-FIG. 2-6L) Representative sagittal high-resolution microcomputedtomography (μCT) images (FIG. 2-6F, left), safranin O/fast greenstaining in low magnification and high magnification images (FIG. 2-6F,middle), CGRP and COX-2 immuno-staining (FIG. 2-6F, right) of the L4/5monkeys' endplates in Vehicle group (Top) or PTH group (Bottom). Scalebars, 200 μm. (FIG. 2-6G-FIG. 2-6J) Quantitative analysis of thepercentage of the total porosity (FIG. 2-6G), the endplate volume (FIG.2-6H), the volume of porosity (FIG. 2-6I) and area of cartilage (FIG.2-6K) in endplates. (FIG. 2-6K-FIG. 2-6L) Quantitative analysis of thepercentage of CGRP nerve fibers (FIG. 2-6K) and COX-2+ cells (FIG. 2-6L)in the endplates of Vehicle group or PTH group. Data are shown asmean±s.d. *p<0.05, **p<0.01;

FIG. 2-7A, FIG. 2-7B, FIG. 2-7C, FIG. 2-7D, FIG. 2-7E, and FIG. 2-7Fprovide the information of aging monkey recruited in this study. (FIG.2-7A) Aging rhesus monkeys had been screened by MRI and done thetreatment according to the schedule. (FIG. 2-7B) Representative imagesof Pfirrmann grade 1-5 of nucleus pulposus in monkey. (FIG. 2-7C) Theinformation of number, gender, and weight in recruited aging rhesusmonkeys. (FIG. 2-7D) Blood (Serum) test including PINP, β-CTx, et al. inmonkeys before PTH or Vehicle treatment. (FIG. 2-7E) The weight changeof aging rhesus monkeys with PTH or Vehicle treatment. (FIG. 2-7F) Theserum test including PINP, β-CTx, Phosphorus, ALP, Osterix, Calciumbefore intervention and after 3 m, 6 m PTH or vehicle treatment. Dataare shown as mean±s.d. *p<0.05, **p<0.01. PINP: procollagen type 1 Npropeptide; β-CTx: C-terminal cross-linked telopeptide of type Icollagen; P: Phosphorus; ALP: Alkaline phosphatase: OST: Osterix; Ca:Calcium;

FIG. 3-1A, FIG. 3-1B, FIG. 3-4C, FIG. 3-1D, FIG. 3-4E, FIG. 3-1F, FIG.3-1G, FIG. 3-1H, FIG. 3-1I, and FIG. 3-1J demonstrate that PTH improvesOA pain and joint degeneration after DMM. (FIG. 3-1A) Paw withdrawalthreshold (PWT) was tested at the left hind paw of sham-operated.PTH-treated DMM and vehicle-treated DMM mice at different time point.n=8/group. (FIG. 3-1B) Paw withdrawal threshold was tested by Pressureapplication measurement (PAM) device at the left knee joint ofsham-operated, PTH-treated DMM and vehicle-treated DMM mice. n=8/group.(FIG. 3-4C) Representative images of gait analysis of sham-operated.PTH-treated DMM and vehicle-treated DMM mice. RH=right hind (pink).LH=left hind (green). RF=right front (blue), LF=left front (yellow).(FIG. 3-1D) Quantitative analysis of percentage LH paw intensity, LHarea and LH swing speed relative to RH at 8 weeks after DMM. n=8/group.(FIG. 3-1E) Safranin O-Fast green staining of sagittal sections of tibiamedial compartment, proteoglycan (red) and bone (green). Scale Bar, 500μm. (FIG. 3-1F) OARSI scores at 2, 4- and 8-weeks post-surgery.n=8/group. (FIG. 3-1G, FIG. 3-4H) Immunohistochemical analysis of matrixmetalloproteinase 13⁺ (MMP13, brown) and type X collagen⁺ (Col X, brown)in articular cartilage. scale Bar, 50 sm. (FIG. 3-1I. FIG. 3-1J)Quantitative analysis of MMP13⁺ and Col X cells in articular cartilage.All data are shown as means±standard deviations. n=8/group. *P<0.05,**P<0.01. NS, no significant difference;

FIG. 3-2A, FIG. 3-2B, FIG. 3-2C, FIG. 3-2D, FIG. 3-2E, FIG. 3-2F, FIG.3-2G, FIG. 3-2H, FIG. 3-21 . and FIG. 3-2J demonstrate that sensorynerve innervation in subchondral bone decreased with PTH treatment.(FIG. 3-2A) Immunofluorescence analysis of CGRP⁺ (1 row, green),Substance P⁺ (2^(nd) row, red), P2X3′(3^(th) row, red), NF200⁺ (4^(th)row, red), PIZEO⁺ (5^(th) row, red) sensory nerve fibers and PGP9.5⁺(6^(th) row, red) nerve fibers in tibial subchondral bone after DMM insham-operated, PTH-treated DMM and vehicle-treated DMM mice. DAPI stainsnuclei (blue). Scale Bar, 50 μm. (FIG. 3-2B-FIG. 3-2G) The quantitativeanalysis of the density of CGRP⁺, SP⁺, P2X3⁺, NF200⁺, PIZEO⁺ sensorynerve fibers and PGP9.5⁺ nerve fibers in tibial subchondral bone afterDMM. n=8/group. (FIG. 3-2H) Immunofluorescence analysis of CGRP⁺ (top,green) sensory nerve fibers and PGP9.5⁺ (bottom, red) nerve fibers inthe synoviμm after DMM in sham-operated. PTH-treated and vehicle-treatedDMM mice. DAPI stains nuclei (blue). Scale Bar, 50 μm. (FIG. 3-2I, FIG.3-2J) The quantitative analysis of the density of CGRP⁺, sensory nervefibers and PGP9.5+ nerve fibers in the synoviμm after DMM insham-operated, PTH-treated DMM and vehicle-treated DMM mice. n=8/group.*P<0.05, **P<0.01. NS, no significant difference:

FIG. 3-3A, FIG. 3-3B, FIG. 3-3C, FIG. 3-3D, FIG. 3-3E, FIG. 3-3F, andFIG. 3-3G demonstrate that PTH sustains subchondral bonemicroarchitecture by remodeling. (FIG. 3-3A) Top row: Three-dimensionalhigh-resolution ICT images of tibial subchondral bone medial compartment(sagittal view) at 8 weeks post sham-operated, PTH-treated DMM andvehicle-treated DMM. Scale bar: 1 mm. Bottom row: Immunohistochemicalanalysis of COX2⁺ cells in mouse tibial subchondral bone after DMMsurgery. Scale bar: 50 μm. (FIG. 3-3B-FIG. 3-3E) Quantitative analysisof structural parameters of subchondral bone by CT analysis: thicknessof subchondral bone plates (SBP.Th), trabecular pattern factor (Tb. Pf),structure model index (SMI) and total volume of pore space Po.V (tot).n=8/group. (FIG. 3-3F) The quantitative analysis of COX2⁺ cells in mousetibial subchondral bone. n=8/group. (FIG. 3-3G) Quantitative analysis ofPGE₂ in subchondral bone determined by Elisa. n=8/group. (H) Trichromestaining in tibial subchondral bone sections. Scale bar: 50 μm. (I)Calcein (green) and alizarin red (red) fluorescent double labeling.Scale bar: 50 μm. *P<0.05, **P<0.01;

FIG. 3-4A, FIG. 3-4B, FIG. 3-4C, FIG. 3-4D, FIG. 3-4E, FIG. 3-4F, FIG.3-4G, FIG. 3-4H, FIG. 3-4I, and FIG. 3-4J demonstrate that PTH sustainssubchondral bone remodeling by endocytosis of TGFβIIR. (FIG. 3-4A)Immunofluorescence or immunohistochemical analysis and quantification ofnestin⁺ cells (top, green) and osterix⁺ cells (bottom, brown) in tibialsubchondral bone after sham operation PTH-treated DMM, orvehicle-treated DMM mice. Scale bar: 50 μm. (FIG. 3-4B, FIG. 3-4C) Thequantification of nestin⁺ cells and osterix⁺ cells in tibial subchondralbone in different groups. n=8/group. (FIG. 3-4D, FIG. 3-4F) Theimmunohistochemical analysis and quantification of pSmad2/3⁺ cells(brown) in mouse tibial subchondral bone of sham-operated. PTH-treatedor Vehicle-treated DMM mice. Scale bar, 50 μm; n=8/group. (FIG. 3-4E,FIG. 3-4G) The TRAP staining (pink) and quantitative analysis of TRAP⁺cells in mouse tibial subchondral bone of sham-operated, PTH-treated orVehicle-treated DMM mice. Scale bar: 100 μm. n=8/group. (FIG. 3-4H)Quantitative analysis of active TGFβ in serum determined by Elisa.n=8/group. (FIG. 3-4I) Immunofluorescent analysis of TGFβIIR (green)distribution on mouse BMSC. Actin (red); DAPI stains nuclei (blue) Scalebar, 20 μm. (FIG. 3-4J) Immunofluorescent analysis of pSmad2/3⁺ on mouseBMSC. Scale bar, 50 μm. DAPI stains nuclei (blue). *P<0.05, **P<0.01;

FIG. 3-5A, FIG. 3-5B, FIG. 3-5C, FIG. 3-5D, FIG. 3-5E, FIG. 3-5F, FIG.3-50 , FIG. 3-5H, FIG. 3-51 , FIG. 3-5J, and FIG. 3-5K demonstrate thatdelayed PTH attenuates progressive OA pain and joint degeneration in DMMmodel. (FIG. 3-5A, FIG. 3-5B) PWT at the left hind paw and withdrawalthreshold tested by PAM at left knee joint in sham-operated, PTH-treatedDMM and vehicle-treated DMM mice, starting from 4 weeks to 8 weeks aftersurgery. n=8/group. (FIG. 3-5C) Quantitative analysis of percentage ofLH paw intensity, LH area and LH swing speed relative to RH, based onCatWalk analysis. n=8/group. (FIG. 3-5D) Immunofluorescent analysis ofthe density of CGRP⁺ (top, green) and SP⁺ (bottom, red) sensory nervefibers in tibial subchondral bone of sham-operated, PTH-treated DMM andvehicle-treated DMM mice. Scale bar, 50 μm. (FIG. 3-5E, FIG. 3-5F) Thequantitative analysis of the density of CGRP⁺, SP⁺ sensory nerve fibersin tibial subchondral bone after DMM in sham-operated, PTH-treated DMMand vehicle-treated DMM mice. DAPI stains nuclei (blue). n=8/group.(FIG. 3-5G) Safranin O-Fast green staining of sagittal sections of tibiamedial compartment, proteoglycan (red) and bone (green) and OARSI. Scalebar, 500 μm. n=8/group. (FIG. 3-5H) Three-dimensional high-resolutionPCT images of tibial subchondral bone medial compartment (sagittal view)post sham-operated, PTH-treated DMM and vehicle-treated DMM (top). Scalebar: 1 mm. Immunohistochemical analysis of COX2⁺ cells in mouse tibialsubchondral bone (bottom, brown). Scale bar, 50 μm. (FIG. 3-5I)Quantitative analysis of structural parameters of subchondral bone byμCT analysis: SBP.Th, Th. Pf, SMI and Po.V(tot). n=8/group. (FIG. 3-5J)Immunohistochemical analysis and quantitative analysis of COX2⁺ cells inmouse tibial subchondral bone. Quantitative analysis of PGE₂ insubchondral bone determined by Elisa. n=8/group. (FIG. 3-5K)Immunohistochemical or immunofluorescent analysis and quantitativeanalysis of osterix+(brown) cells in tibial subchondral bone. Scale bar,50 μm. n=8/group. *P<0.05, **P<0.01:

FIG. 3-6A, FIG. 3-6B, FIG. 3-6C, FIG. 3-6D, FIG. 3-6E, FIG. 3-6F, andFIG. 3-6G demonstrate that PTH-induced OA pain relief inhibited by PTH1Rknockout on Nestin⁺ MSCs. (FIG. 3-6A, FIG. 3-6B) PWT at the left hindpaw and withdrawal threshold tested by PAM at left knee joint insham-operated, PTH-treated DMM and vehicle-treated DMM PTH1^(−/−) andPTH1R^(+/+) mice. n=8/group. (FIG. 3-6C) Quantitative analysis of LH pawintensity. LH area and LH swing speed relative to RH in sham-operated,PTH-treated DMM and vehicle-treated DMM PTH1 R^(−/−) and PTH1R^(+/+)mice, based on CatWalk analysis. n=8/group. (FIG. 3-6D)Immunofluorescent analysis of CGRP⁺ (1^(st) row, green), Substance P⁺(2^(nd) row, red), P2X3⁺ (3^(rd) row, red), NF200⁺ (4^(th) row, red),PIZEO⁺ (5^(th) row, red) sensory nerve fibers and PGP9.5⁺ (6^(th) row,red) nerve fibers in tibial subchondral bone of PTH-treated orvehicle-treated PTH1R^(−/−) and PTH1R^(+/+) mice. Scale bar, 50 μm.(FIG. 3-6E) The quantitative analysis of the density of CGRP⁺, SP⁺,P2X3⁺, NF200⁺, PIZEO⁺ sensory nerve fibers and PGP9.5⁺ nerve fibers intibial subchondral bone of sham-operated, PTH-treated DMM andvehicle-treated DMM PTH1R⁺ and PTH1R^(+/+) mice. n=8/group. (FIG. 3-6F,FIG. 3-6G) Immunofluorescent and quantitative analysis of CGRP⁺ (top,green) sensory nerve fibers and PGP9.5⁺ (bottom, red) nerve fibers ofsham-operated, PTH-treated DMM and vehicle-treated DMM PTH1R^(−/−) andPTH1R^(+/+) mice. Scale bar, 50 μm. n=8/group. *P<0.05, **P<0.01. NS, nosignificant difference, and

FIG. 3-7A. FIG. 3-713 . FIG. 3-7C, FIG. 3-7D, FIG. 3-7E, FIG. 3-7F, andFIG. 3-7G. and FIG. 3-7H demonstrate that PTH-induced bone remodelinginhibited by PTH1R knockout on Nestin⁺ MSCs. (FIG. 3-7A) Top: SafraninO-Fast green staining of sagittal sections of tibia medial compartment,proteoglycan (red) and bone (green). Scale bar, 500 μm; Middle.three-dimensional high-resolution μCT images of tibial subchondral bonemedial compartment. Scale bar 1 mm: Bottom: Immunohistochemical analysisof COX2⁺ (brown) cells in mouse tibial subchondral bone. Scale bar, 50μm: (FIG. 3-7B) OARSI score. N=8/group (FIG. 3-7C) Quantitative analysisof structural parameters of subchondral bone by μCT analysis: SBP.Th,Th. Pf, SMI and Po.V(tot). n=8/group. (FIG. 3-7D) The quantitativeanalysis of COX2⁺ cells in mouse tibial subchondral bone. n=8/group.(FIG. 3-7E) Quantitative analysis of PGE2 in subchondral bone determinedby Elisa. n=8/group. (FIG. 3-7F) Immunohistochemical analysis andquantification of pSmad2/3⁺ cell in subchondral bone marrow. Scale bar,50 μm; n=8/group. (FIG. 3-7G, FIG. 3-7H) The Immunofluorescent orimmunohistochemical analysis and quantification of nestin+ cells (green)and osterix⁺ (brown) cells in tibial subchondral bone. Scale bar, 50 μm;n=8/group. *P<0.05, **P<0.01. NS, no significant difference.

DETAILED DESCRIPTION

The presently disclosed subject matter now will be described more fullyhereinafter with reference to the accompanying Figures, in which some,but not all embodiments of the inventions are shown. Like numbers referto like elements throughout. The presently disclosed subject matter maybe embodied in many different forms and should not be construed aslimited to the embodiments set forth herein: rather, these embodimentsare provided so that this disclosure will satisfy applicable legalrequirements. Indeed. many modifications and other embodiments of thepresently disclosed subject matter set forth herein will come to mind toone skilled in the art to which the presently disclosed subject matterpertains having the benefit of the teachings presented in the foregoingdescriptions and the associated Figures. Therefore, it is to beunderstood that the presently disclosed subject matter is not to belimited to the specific embodiments disclosed and that modifications andother embodiments are intended to be included within the scope of theappended claims.

I. Parathyroid Hormone Attenuates Low Back Pain and Osteoarthritic Pain

In some embodiments, the presently disclosed subject matter provides amethod for treating low back pain (LBP) and/or osteoarthritic pain in asubject in need of treatment thereof, the method comprisingadministering to the subject a composition comprising a recombinantparathyroid hormone (PTH) and a pharmaceutically acceptable carrier.

In certain embodiments, the low back pain comprises a nonspecific lowback pain.

In certain embodiments, the administering of the recombinant parathyroidhormone (PTH) inhibits osteoclast activity-induced sensory innervationin a vertebral endplate of the subject.

As used herein, the term “inhibit,” and grammatical derivations thereof,refers to the ability of a presently disclosed compound, e.g., arecombinant parathyroid hormone (PTH), to block, partially block,interfere, decrease. or reduce, for example, osteoclast activity-inducedsensory innervation in a vertebral endplate. Thus, one of ordinary skillin the art would appreciate that the term “inhibit” encompasses acomplete and/or partial decrease in an activity, e.g., a decrease by atleast 10%. in some embodiments, a decrease by at least 20%, 30%, 50%,75%, 95%, 98%, and up to and including 100%.

In particular embodiments, the administering of the recombinantparathyroid hormone (PTH) treats the osteoarthritic pain by one or moreof inhibition of nerve innervation, inhibition of subchondral bonedeterioration, inhibition of articular cartilage degeneration.attenuation of joint degeneration, decelerating subchondral bonedeterioration, and sustaining of subchondral bone microarchitecture byremodeling.

In other embodiments, the administering of the recombinant parathyroidhormone (PTH) increases the intervertebral disc (IV D) space bydecreasing the volume and porosity of sclerotic endplates.

In yet other embodiments, the administering of the recombinantparathyroid hormone (PTH) prevents endplate remodeling and sclerosis.

In even yet other embodiments, the administering of the recombinantparathyroid hormone (PTH) reduces sensory nerve fibers.

In other embodiments, the administering of the recombinant parathyroidhormone (PTH) reduces the porosity of sclerotic endplates.

In some embodiments, the method further comprises administering at leastone other agent in combination with the administering of the recombinantparathyroid hormone (PTH).

In certain embodiments, the at least one other agent is selected fromthe group consisting of paracetamol, an opioid, a non-steroidalanti-inflammatory drug, a skeletal muscle relaxant, a triptan, anα2-agonist, a local anesthetic, a tricyclic antidepressant, abenzodiazepine, a steroid, a visco supplement, and combinations thereof.

In particular embodiments, the low back pain is associated with one ormore of spine degeneration, lumbar disc herniation (LDH), scoliosis,cancer, and an infection.

In some embodiments, the recombinant PTH comprises a full-length PTHprotein or a fragment of PTH. In particular embodiments, the recombinantparathyroid hormone comprises teriparatide (PTH(I-34′)). In otherembodiments, the recombinant parathyroid hormone comprises an intactparathyroid hormone (iPTH).

In certain embodiments, the composition is administered to the subjectat least once a day.

In other embodiments, the presently disclosed subject matter providesthe use of a recombinant parathyroid hormone to treat low back pain(LBP) or osteoarthritic pain in a subject in need thereof.

As used herein, the term “treating” can include reversing, alleviating,inhibiting the progression of, preventing or reducing the likelihood ofthe disease, disorder, or condition to which such term applies, or oneor more symptoms or manifestations of such disease, disorder orcondition. Preventing refers to causing a disease, disorder, condition,or symptom or manifestation of such, or worsening of the severity ofsuch, not to occur. Accordingly, the presently disclosed compounds canbe administered prophylactically to prevent or reduce the incidence orrecurrence of the disease, disorder, or condition.

The “subject” treated by the presently disclosed methods in their manyembodiments is desirably a human subject, although it is to beunderstood that the methods described herein are effective with respectto all vertebrate species, which are intended to be included in the term“subject.” Accordingly, a “subject” can include a human subject formedical purposes. such as for the treatment of an existing condition ordisease or the prophylactic treatment for preventing the onset of acondition or disease, or an animal subject for medical, veterinarypurposes, or developmental purposes. Suitable animal subjects includemammals including, but not limited to, primates, e.g., humans, monkeys,apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines,e.g., sheep and the like; caprines, e.g., goats and the like; porcines,e.g., pigs, hogs, and the like: equines, e.g., horses. donkeys, zebras,and the like; felines, including wild and domestic cats; canines,including dogs; lagomorphs, including rabbits, hares, and the like; androdents, including mice, rats, and the like. An animal may be atransgenic animal. In some embodiments, the subject is a humanincluding, but not limited to, fetal, neonatal, infant, juvenile, andadult subjects. Further, a “subject” can include a patient afflictedwith or suspected of being afflicted with a condition or disease. Thus,the terms “subject” and “patient” are used interchangeably herein. Theterm “subject” also refers to an organism, tissue, cell, or collectionof cells from a subject.

In general, the “effective amount” of an active agent or drug deliverydevice refers to the amount necessary to elicit the desired biologicalresponse. As will be appreciated by those of ordinary skill in this art,the effective amount of an agent or device may vary depending on suchfactors as the desired biological endpoint, the agent to be delivered,the makeup of the pharmaceutical composition, the target tissue, and thelike.

The term “combination” is used in its broadest sense and means that asubject is administered at least two agents, more particularly arecombinant parathyroid hormone (PTH) and at least one other agent. Insome embodiments, the at least one other agent is selected from thegroup consisting of paracetamol, opioids, non-steroidalanti-inflammatory drugs (NSAIDs, including COX-2 inhibitors), andskeletal muscle relaxants. In other embodiments, the at least one otheragent is selected from the group consisting of triptans, α2-agonists,and local anesthetics. In chronic cases, the at least one other agentsis a tricyclic antidepressant and/or a benzodiazepine.

More particularly, the term “in combination” refers to the concomitantadministration of two (or more) active agents for the treatment of a,e.g., single disease state. As used herein, the active agents may becombined and administered in a single dosage form, may be administeredas separate dosage forms at the same time, or may be administered asseparate dosage forms that are administered alternately or sequentiallyon the same or separate days. In one embodiment of the presentlydisclosed subject matter. the active agents are combined andadministered in a single dosage form. In another embodiment, the activeagents are administered in separate dosage forms (e.g., wherein it isdesirable to vary the amount of one but not the other). The singledosage form may include additional active agents for the treatment ofthe disease state.

Further, the recombinant parathyroid hormone (PTH) described herein canbe administered alone or in combination with adjuvants that enhancestability of the recombinant parathyroid hormone (PTH), alone or incombination with one or more antibacterial agents, facilitateadministration of pharmaceutical compositions containing them in certainembodiments, provide increased dissolution or dispersion, increaseinhibitory activity, provide adjunct therapy, and the like, includingother active ingredients. Advantageously, such combination therapiesutilize lower dosages of the conventional therapeutics, thus avoidingpossible toxicity and adverse side effects incurred when those agentsare used as monotherapies.

The timing of administration of a recombinant parathyroid hormone (PTH)and at least one additional therapeutic agent can be varied so long asthe beneficial effects of the combination of these agents are achieved.Accordingly, the phrase “in combination with” refers to theadministration of a recombinant parathyroid hormone (PTH) and at leastone additional therapeutic agent either simultaneously, sequentially, ora combination thereof. Therefore, a subject administered a combinationof a recombinant parathyroid hormone (PTH) and at least one additionaltherapeutic agent can receive a recombinant parathyroid hormone (PTH)and at least one additional therapeutic agent at the same time (i.e.,simultaneously) or at different times (i.e., sequentially, in eitherorder, on the same day or on different days), so long as the effect ofthe combination of both agents is achieved in the subject.

When administered sequentially, the agents can be administered within 1,5, 10, 30, 60, 120, 180, 240 minutes or longer of one another. In otherembodiments, agents administered sequentially, can be administeredwithin 1, 5, 10, 15, 20 or more days of one another. Where therecombinant parathyroid hormone (PTH) and at least one additionaltherapeutic agent are administered simultaneously, they can beadministered to the subject as separate pharmaceutical compositions,each comprising either a recombinant parathyroid hormone (PTH) or atleast one additional therapeutic agent, or they can be administered to asubject as a single pharmaceutical composition comprising both agents.

When administered in combination, the effective concentration of each ofthe agents to elicit a particular biological response may be less thanthe effective concentration of each agent when administered alone,thereby allowing a reduction in the dose of one or more of the agentsrelative to the dose that would be needed if the agent was administeredas a single agent. The effects of multiple agents may, but need not be,additive or synergistic. The agents may be administered multiple times.

In some embodiments, when administered in combination, the two or moreagents can have a synergistic effect. As used herein, the terms“synergy,” “synergistic,” “synergistically” and derivations thereof,such as in a “synergistic effect” or a “synergistic combination” or a“synergistic composition” refer to circumstances under which thebiological activity of a combination of a recombinant parathyroidhormone (PTH) and at least one additional therapeutic agent is greaterthan the sum of the biological activities of the respective agents whenadministered individually.

Synergy can be expressed in terms of a “Synergy Index (SI),” whichgenerally can be determined by the method described by F. C. Kull etal., Applied Microbiology 9, 538 (1961), from the ratio determined by:

Q _(a) /Q _(A) +Q _(b) /Q _(B)=Synergy Index (SI)

wherein:

Q_(A) is the concentration of a component A. acting alone, whichproduced an end point in relation to component A;

-   -   Q_(a) is the concentration of component A, in a mixture, which        produced an end point;    -   Q_(B) is the concentration of a component B. acting alone, which        produced an end point in relation to component B: and    -   Q_(b) is the concentration of component B, in a mixture, which        produced an end point.

Generally. when the sum of Q_(a)/Q_(A) and Q_(b)/Q_(B) is greater thanone, antagonism is indicated. When the sum is equal to one, additivityis indicated. When the sum is less than one. synergism is demonstrated.The lower the SI, the greater the synergy shown by that particularmixture. Thus, a “synergistic combination” has an activity higher thatwhat can be expected based on the observed activities of the individualcomponents when used alone. Further, a “synergistically effectiveamount” of a component refers to the amount of the component necessaryto elicit a synergistic effect in, for example, another therapeuticagent present in the composition.

Pharmaceutical Compositions and Administration

In another aspect, the present disclosure provides a pharmaceuticalcomposition including a recombinant parathyroid hormone (PTH) alone orin combination with one or more additional therapeutic agents inadmixture with a pharmaceutically acceptable excipient. One of skill inthe art will recognize that the pharmaceutical compositions include thepharmaceutically acceptable salts of the compounds described above.

In therapeutic and/or diagnostic applications, the compounds of thedisclosure can be formulated for a variety of modes of administration,including systemic and topical or localized administration. Techniquesand formulations generally may be found in Remington: The Science andPractice of Pharmacy (20^(th) ed.) Lippincott, Williams & Wilkins(2000).

Depending on the specific conditions being treated, such agents may beformulated into liquid or solid dosage forms and administeredsystemically or locally. The agents may be delivered, for example, in atimed- or sustained-slow release form as is known to those skilled inthe art. Techniques for formulation and administration may be found inRemington: The Science and Practice of Pharmacy (20^(th) ed.)Lippincott, Williams & Wilkins (2000). Suitable routes may include oral,buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal,transmucosal, nasal or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intra-articular, intra-sternal, intra-synovial, intra-hepatic,intralesional, intracranial, intraperitoneal, intranasal, or intraocularinjections or other modes of delivery.

For injection, the agents of the disclosure may be formulated anddiluted in aqueous solutions. such as in physiologically compatiblebuffers such as Hank's solution, Ringer's solution, or physiologicalsaline buffer. For such transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

Use of pharmaceutically acceptable inert carriers to formulate thecompounds herein disclosed for the practice of the disclosure intodosages suitable for systemic administration is within the scope of thedisclosure. With proper choice of carrier and suitable manufacturingpractice, the compositions of the present disclosure, in particular.those formulated as solutions. may be administered parenterally, such asby intravenous injection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe disclosure to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya subject (e.g., patient) to be treated.

For nasal or inhalation delivery, the agents of the disclosure also maybe formulated by methods known to those of skill in the art, and mayinclude, for example, but not limited to, examples of solubilizing.diluting, or dispersing substances. such as saline; preservatives, suchas benzyl alcohol: absorption promoters; and fluorocarbons.

Pharmaceutical compositions suitable for use in the present disclosureinclude compositions wherein the active ingredients are contained in aneffective amount to achieve its intended purpose. Determination of theeffective amounts is well within the capability of those skilled in theart, especially in light of the detailed disclosure provided herein.Generally, the compounds according to the disclosure are effective overa wide dosage range. For example, in the treatment of adult humans,dosages from 0.01 to 1000 mg, from 0.5 to 100 mg. from 1 to 50 mg perday. and from 5 to 40 mg per day are examples of dosages that may beused. A non-limiting dosage is 10 to 30 mg per day. The exact dosage w %ill depend upon the route of administration, the form in which thecompound is administered, the subject to be treated, the body weight ofthe subject to be treated, the bioavailability of the compound(s), theadsorption, distribution, metabolism, and excretion (ADME) toxicity ofthe compound(s), and the preference and experience of the attendingphysician.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Thepreparations formulated for oral administration may be in the form oftablets, dragees, capsules, or solutions.

Pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with solid excipients, optionally grinding aresulting mixture, and processing the mixture of granules. after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations, for example, maize starch, wheat starch, rice starch,potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (CMC),and/or polyvinylpyrrolidone (PVP: povidone). If desired. disintegratingagents may be added, such as the cross-linked polyvinylpyrrolidone,agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used. which may optionally containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol (PEG), and/or titanium dioxide, lacquer solutions, and suitableorganic solvents or solvent mixtures. Dye-stuffs or pigments may beadded to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin, and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols (PEGs). In addition, stabilizers may be added.

Definitions

Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation. Unlessotherwise defined, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this presently described subject matter belongs.

Following long-standing patent law convention, the terms “a,” “an,” and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a subject” includes aplurality of subjects, unless the context clearly is to the contrary(e.g., a plurality of subjects), and so forth.

Throughout this specification and the claims, the terms “comprise,”“comprises,” and “comprising” are used in a non-exclusive sense, exceptwhere the context requires otherwise. Likewise, the term “include” andits grammatical variants are intended to be non-limiting, such thatrecitation of items in a list is not to the exclusion of other likeitems that can be substituted or added to the listed items.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing amounts, sizes, dimensions,proportions, shapes, formulations, parameters, percentages, quantities,characteristics, and other numerical values used in the specificationand claims, are to be understood as being modified in all instances bythe term “about” even though the term “about” may not expressly appearwith the value, amount or range. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the followingspecification and attached claims are not and need not be exact, but maybe approximate and/or larger or smaller as desired, reflectingtolerances, conversion factors, rounding off, measurement error and thelike, and other factors known to those of skill in the art depending onthe desired properties sought to be obtained by the presently disclosedsubject matter. For example, the term “about,” when referring to a valuecan be meant to encompass variations of, in some embodiments, +100% insome embodiments±50%, in some embodiments±20%, in some embodiments±10%,in some embodiments f 5%, in some embodiments ±1%, in someembodiments±0.5%, and in some embodiments t 0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethods or employ the disclosed compositions.

Further, the term “about” when used in connection with one or morenumbers or numerical ranges, should be understood to refer to all suchnumbers, including all numbers in a range and modifies that range byextending the boundaries above and below the numerical values set forth.The recitation of numerical ranges by endpoints includes all numbers,e.g., whole integers, including fractions thereof, subsumed within thatrange (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5,as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like)and any range within that range.

EXAMPLES

The following Examples have been included to provide guidance to one ofordinary skill in the art for practicing representative embodiments ofthe presently disclosed subject matter. In light of the presentdisclosure and the general level of skill in the art, those of skill canappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter. The synthetic descriptions and specific examples thatfollow are only intended for the purposes of illustration, and are notto be construed as limiting in any manner to make compounds of thedisclosure by other methods.

Example 1 Sensory Innervation in Porous Endplates by Netrin-1 fromOsteoclasts Mediates PGE2-induced Spinal Hypersensitivity in Mice 1.1Overview

Spinal pain is a major clinical problem. Its origins and underlyingmechanisms, however, remain unclear. In some embodiments, the presentlydisclosed subject matter provides that in mice, osteoclasts inducesensory innervation in the porous endplates, which contributes spinalhypersensitivity in mice. Sensory innervation of the porous areas ofsclerotic endplates in mice was confirmed. Lumbar spine instability(LSI), or aging, induces spinal hypersensitivity in mice. In theseconditions, elevated levels of PGE2, which activate sensory nervesleading to sodium influx through Na_(v)1.8 channels, were observed.Further, knockout of PGE2 receptor 4 in sensory nerves significantlyreduced spinal hypersensitivity. Inhibition of osteoclast formation byknockout Rankl in the osteocytes significantly inhibited LSI-inducedporosity of endplates, sensory innervation, and spinal hypersensitivity.Knockout of Netrin-1 in osteoclasts abrogates sensory innervation intoporous endplates and spinal hypersensitivity. These findings suggestthat osteoclast-initiated porosity of endplates and sensory innervationare potential therapeutic targets for spinal pain.

1.2 Background

Low back pain (LBP) is a common health problem, which most people (80%)experience at some point, especially in older adults. Rubin, 2007:Hartvigsen et al., 2006; Hartvigsen et al., 2004: and Global, regional,and national incidence, prevalence, and years lived with disability for310 diseases and injuries, 1990-2015: a systematic analysis for theGlobal Burden of Disease Study 2015.

In the United States alone, the direct and indirect costs associatedwith LBP surpass $90 billion per year, with similar adjusted rates inother countries. Samartzis and Grivas, 2017. 90% of LBP is nonspecificLBP, which has no apparent pathoanatomical cause. Krismer and vanTulder, 2007: Koes et al., 2006. Several lumbar structures, such asintervertebral disc, facet joints, are plausible sources of nonspecificLBP, but the pain cannot be reliably attributed to those structures byclinical tests. Hancock et al., 2007; Maher et al., 2017; and Hartvigsenet al., 2018. Importantly, intervertebral disc (IVD) degeneration isfrequently observed in asymptomatic patients, indicating that discdegeneration, per se, is not painful in some patients. Hurn andKarppinen, 2004: Borenstein et al., 2001. Hence, identifying the sourceof LBP and related mechanisms is essential to develop effectivetreatments for LBP.

The positive association between vertebral endplate signal changes(i.e., Modic changes) and LBP has been shown by magnetic resonanceimaging (MRI) examination. Rahme and Moussa, 2008; Luoma et al., 2016.Modic changes are common MRI findings in patients with nonspecific LBP.It is believed to be a factor independently associated with theincreased risk of LBP. Jensen et al., 2008: Maatta et al., 2015: andJensen et al., 2014. The size of Modic change lesions positivelycorrelates with LBP. Jarvinen et al., 2015. Histological analysisfurther showed that the presence of endplate lesions was associated withLBP. Wang et al., 2012. During aging, endplates undergo ossificationwith elevated osteoclasts and become porous. Bian et al., 2016: Bian etal., 2017; Rodriguez et al., 2012; and Papadakis et al., 2011.Histological and micro CT analysis further revealed that scleroticendplates are highly porous. Rodriguez et al., 2012. Progressively moreporous endplates with narrowed IVD space are characteristics of spinaldegeneration. Rodriguez et al., 2012; Taher et al., 2012. Since pain isproduced by nociceptors, LBP may be caused by sensory innervation intoendplates. Moreover, nerve density was higher in porous endplates thanin normal endplates or degenerative nucleus pulposus. Fields et al.,2014. Zoledronic acid and denosumab, drugs that inhibit osteoclastactivities, have shown analgesic effects in patients with Modic changesassociated LBP, Cai et al., 2018; Koivisto et al., 2014, with theimplication of a potential role of osteoclast activity in sensory nerveinnervation.

Prostaglandin E2 (PGE2) is an inflammatory mediator released at thefocal inflamed tissue and a neuromodulator that alters neuronalexcitability. Four types of G-protein-coupled EP receptors (EP1-EP4)mediate the functions of PGE2. EP4 receptor is considered the primarymediator of PGE2-evoked inflammatory pain hypersensitivity andsensitization of sensory neurons. Lin et al., 2006: Southall and Vasko,2001. It was recently reported that PGE2, produced from arachidonic acidby the enzymatic activity of cyclooxygenase 2 (COX2), during boneremodeling activates PGE2 receptor 4 (EP4) in CGRP⁺ sensory nerves totune down sympathetic tones further inducing osteoblasticdifferentiation of MSCs. Chen et al, 2019. Specific EP4 receptorantagonists could reduce acute and chronic pain, includingosteoarthritis pain. Lin et al., 2006; Clark et al., 2008: Kirkby Shawet al., 2016; Nakao et al., 2007. The tetrodotoxin-resistant (TX-R)sodium channel Na_(v)1.8 is a potential drug target for pain. Na_(v)1.8is expressed primarily in small and medium-sized dorsal root ganglion(DRG) neurons and their fibers. Janis et al., 2007: Coggeshall et al.,2004: and Schuelert and McDougall, 2012. PGE2 could modulate the TTX-Rsodium current in DRG neurons and promote Na_(v)1.8 trafficking to thecell surface. Liu et al., 2010; England et al., 1996.

One aspect of the presently disclosed study is to demonstrate thatosteoclasts initiate porosity of endplates with sensory innervation intoporous areas. The presently disclosed data show that CalcitoninGene-Related Peptide positive (CGRP⁺) nociceptive nerve fibers and bloodvessels were increased in the cavities of sclerotic endplates. Theelevated PGE2 in porous endplates induces sodium influx into the cellsto stimulate sensory nerves that leads to spinal pain. Inhibition ofosteoclast activity attenuated sensory innervation in porous endplatesand pain behavior.

1.3 Results 1.3.1 Development of Hyperalgesia in LSI and Aged MouseModels

Lumbar spine instability (LSI) was established in mice as a spinedegeneration model for spine pain behavior testing. Bian et al., 2016;Ariga et al., 2001; and Miyamoto et al., 2001. The vocalizationthreshold was first measured in response to force applied on the L4/L5disc region. Pressure tolerance decreased significantly at 4, 8, and 12weeks after LSI surgery relative to mice that underwent sham surgery(FIG. 1-1A), indicating the development of low back pressurehyperalgesia. Then, spontaneous activity was monitored to indicate thepotential effect of spinal pain, including distance traveled, maximumspeed of movement, mean speed of movement, and active time per 24 hours,though they are not specific for spinal pain behaviors. The resultsrevealed that each measure of spontaneous activity decreasedsignificantly at 4 and 8 weeks after LSI surgery relative to shamsurgery (FIG. 1-1B-FIG. 1-1E). Moreover, mechanical hyperalgesia of thehind paw as referred pain was assessed by performing von Frey analysis,as a secondary indicator of symptomatic LBP. Paw withdraw frequencyincreased significantly from 2 to 12 weeks after LSI surgery (FIG. 1-1F,FIG. 1-1G). However, no response to the straight leg raising test wasobserved when recording the number of vocalizations during 5 legstretch-and-lifts in either LSI or sham surgery mice. This observationindicates that nerve root compression is not involved in thehyperalgesia developed after LSI surgery. The results of these painbehavior tests suggest that spine instability induces the development ofhyperalgesia.

In parallel, symptomatic LBP during aging in these behavior tests wasevaluated. Similarly, the threshold of pressure tolerance (FIG. 1-1H)and spontaneous activity (FIG. 1-1I-FIG. 1-1K) decreased significantlyin aged mice (age 20 months) relative to young mice (age 3 months). Themechanical hyperalgesia of the hind paw increased significantly in agedmice relative to young mice (FIG. 1-1L, 1-1M). Together, these dataindicate that, as in the LSI mouse model, aging also induces spinehyperalgesia.

1.3.2 Sensory Innervation in Endplates in LS1 and Aged Mouse Models

An increase in osteoclasts at the onset of endplate sclerosis waspreviously shown. Bian et al., 2016. Therefore, the potential role ofosteoclasts in the sensory innervation of endplates was evaluated inthis study. Tartrate-resistant acid phosphatase (TRAP) stainingdemonstrated that the number of TRAP⁺ osteoclasts in endplates increasedsignificantly at 2 weeks after LSI and remained at a high level until 8weeks after LSI surgery (FIG. 1-2A, FIG. 1-2B). Large bone marrowcavities were generated in sclerotic endplates by osteoclastic boneresorption in mice after LSI surgery, whereas, the cartilaginousendplates were maintained in sham surgery mice (FIG. 1-2A).Immunofluorescent staining revealed that the significant increase ofCGRP, the marker of peptidergic nociceptive C nerve fibers in the porousendplates, began at 2 weeks and continued to increase until 8 weeksafter LSI surgery (FIG. 1-2C, FIG. 1-2D), but there were no detectableCGRP⁺ nerves in the endplates of sham surgery mice (FIG. 1-2C, FIG.1-2D). Interestingly, the nociceptive nerve fibers were localizedprimarily adjacent to the bone surface (FIG. 1-2C). The co-staining ofPGP9.5, the broad marker of nerve fibers with CGRP, further validatedthe nociceptive innervation of the endplates after LSI surgery (FIG.1-2E). However, the nonpeptidergic subtype of IB4⁺ C nerve fibers wasnot detected in either LSI or sham surgery mice (FIG. 1-2F), suggestingCGRP⁺ nerve fibers as the primary nociceptive C nerve fibers in theendplates. Importantly. CD3⁺IEMCN⁺ blood vessels were also growing intothe porous endplates after LSI surgery, along with the sensoryinnervation of the endplates (FIG. 1-10A, FIG. 1-10B).

To determine whether spine degeneration during aging could inducesclerosis and sensory innervation in vertebral endplates, the caudalendplates of L4/5 from aged mice and young mice were analyzed. Theporosity of endplates in aged mice increased significantly relative toyoung mice, as determined by 3-dimensional microcomputed tomography(μCT) analysis (FIG. 1-3A-FIG. 1-3C). The μCT analysis of vertebraltrabecular bone demonstrated that the trabecular bone volume/totalvolume (BV/TV) and trabecular bone number (Tb.N) of L5 vertebraedecreased significantly in 20-month-old mice relative to 3-month-oldmice, while the trabecular bone thickness (Tb.Th) and trabecular boneseparation distribution (Tb.Sp) did not change significantly (FIG.1-11A-FIG. 1-11E) Safranin O and fast green staining demonstrated thatthe green-stained bone matrix surrounded the cavities in endplates ofaged mice (FIG. 1-3D, top and middle), suggesting endochondralossification. Endplate scores, which are a histologic assessment ofpathological changes such as bony sclerosis, structure disorganization,and neovascularization, were significantly higher in aged mice than thatin young mice (FIG. 1-3E). Interestingly, high levels of TRAP⁺osteoclasts were observed in the endplates of aged mice, whereas TRAP⁺osteoclasts were rarely detected in the endplates of young mice (FIG.1-3C, bottom and FIG. 1-3F). Immunostaining of CGRP showed increasedaberrant innervation of peptidergic nociceptive C nerve fibers in theporous endplates of aged mice (FIG. 1-3G and FIG. 1-3H) The co-stainingof PGP9.5 and CGRP further confirmed that the endplates were innervatedby nociceptive nerve fibers (FIG. 1-3I). Similar to the findings in LSImice, CD31⁺EMCN⁺ blood vessels were detected in the endplates, alongwith CGRP⁺ nerve fibers during aging (FIG. 1-12A, FIG. 1-12B),indicating active ossification of the endplates.

To examine the potential involvement of sclerosis and sensoryinnervation of the endplates with pain behavior, the pathologicalchanges in the endplates of the lower lumbar spines from patients withor without LBP history was evaluated. Severe endplate lesions wereobserved in patients with a history of frequent LBP, whereas thecartilaginous structure was preserved in patients without a history offrequent LBP, despite disc herniation (FIG. 1-13A). The increasedendplate scores were also observed in patients with a history offrequent LBP (FIG. 1-13B). The patients with the history of frequentLBP, however, are older than the ones without the history of frequentLBP (Table 1).

TABLE 1 Information for the human samples No-Low Back Pain Frequent LowBack Pain Sample Size 4 9 Age (Years) 28.3 ± 3.5 56.6 ± 5.4 Sex(Male/Female) 3/1 5/4 Body Mass Index 22.5 ± 1.9 23.9 ± 2.4 (BMI) DiscLevel L4/5(3)/L5/S1(1) L3/4(2)/L4/5(4)/L5/S1(3) Endplate lesions N/A 8Pfirrmann grading 1/3 4/5 (grade 3/grade 4)

TRAP staining showed that abundant TRAP⁺ osteoclasts localized at thebone surface in the sclerotic endplates (FIG. 1-13C). Immunofluorescencestaining revealed that CGRP⁺PGP9.5⁺ nociceptive nerve fibers grown intothe porous areas of sclerotic endplates of patients with LBP history(FIG. 1-13D). These results suggest that sensory innervation insclerotic endplates is potentially related to spinal pain behavior.

1.3.3 Retrograde and Anterograde Tracing of Sensory Innervation

To demonstrate CGRP⁺ sensory innervation in endplates during spinedegeneration, a retrograde tracing experiment in both LSI and aged micewas conducted. The red fluorescent tracer, Dil, was injected in the leftpart of the caudal endplates of L4/5 in mice at 8 weeks after LSIsurgery (FIG. 1-4A). The T12-L6 dorsal root ganglions (DRGs) in bothsides were harvested at 1 week after injection to calculate the numberof Dil⁺ neurons. It was observed that Dil was retrograded mainly to theleft T13-L3 DRGs, especially the left L1 and L2 DRGs in LSI mice,whereas no Dil⁺ neurons were detected in the T12-L6 DRGs of sham surgerymice (FIG. 1-4B, FIG. 1-4C). Immunofluorescent staining of the DRGsections demonstrated that Dil in the left L1 and L2 DRGs wasco-localized mainly with CGRP⁺ but not IB4⁺ neurons in LSI mice (FIG.14D, FIG. 1-4E).

The anterograde tracing experiment was performed by labeling the L1 andL2 DRG neurons in both sides with injection of Dil at 8 weeks after LSIor sham surgery. Abundant Dil-labeled sensory nerves were seen in theporous areas of endplates of L4/5 in LSI mice, but not in sham surgerymice (FIG. 1-4F, FIG. 1-4G). Similarly, the innervation of Dil-labeledsensory nerves in the porous areas of endplates of aged mice was alsoobserved in the anterograde tracing experiment (FIG. 1-4H, FIG. 1-4I).Taken together, these findings suggest nociceptive innervation in thesclerotic endplates of LSI and aged mice.

1.3.4 PGE2′EP4 Signaling Mediates Spinal Hypersensitivity

To elucidate the signaling mechanism of endplate sclerosis-mediated painbehavior, the expression of several inflammatory cytokines in theendplates of LSI and sham surgery mice was examined by usingquantitative real-time polymerase chain reaction (qRT-PCR). It was foundthat messenger ribonucleic acid levels of prostaglandin E synthase(PGES), cox2, interleukin (IL)-1β, IL-17, IL-2, and tumor necrosisfactor (TNF)-α increased significantly in the lumbar endplates at 4weeks after LSI relative to sham surgery, especially PGES (FIG. 1-5A).Immunostaining further confirmed a significant increase in cox2 in theendplates at 4 and 8 weeks after LSI surgery (FIG. 1-5B, top). Theincreases of PGES, cox2, and IL-10 can contribute to the synthesis ofPGE2 in the endplates, which was validated by immunostaining (FIG. 1-5B,bottom) and enzyme-linked immunoabsorbent assay (ELISA) (FIG. 1-5C). Theincrease of PGE2 in endplates peaked at 4 weeks and remained high at 8and 12 weeks after LSI surgery (FIG. 1-5C). To explore the potentialsource of PGE2 in porous endplates, the co-immunostaining for cox2 withF4/80, cox2 with osteocalcin (OCN), and cox2 with TRAP were conductedrespectively. The results demonstrated that the COX2 was co-localizedwith F4/80⁺, some OCN⁺, and a few TRAP⁺ cells (FIG. 1-14 ). These datashowed that the accumulated PGE2 in porous endplates was derived fromdifferent types of cell. Immunostaining showed that EP4 was expressed innewly innervated CGRP⁺ nerve endings in the endplates of LSI mice (FIG.1-5D). Notably, the proportion of CGRP⁺EP4⁺ neurons relative to CGRP⁺neurons in L2 DRGs was significantly greater in LSI mice relative tosham surgery mice (FIG. 1-5E, FIG. 1-5F). Interestingly, the sodiumchannel Na_(v) 1.8 was also expressed in newly innervated CGRP⁺ nerveending in the endplates of LSI mice, as demonstrated by immunostaining(FIG. 1-5G). Moreover, the proportion of CGRP⁺Na_(v)1.8⁺ neuronsrelative to CGRP⁺ neurons in L2 DRGs increased significantly at 4 and 8weeks after LSI surgery (FIG. 1-5H, FIG. 1-5I).

To examine the potential role of PGE2/EP4 in pain transduction, asensory neuron specific EP4 knockout mice (Avil^(Cre); EP4^(flox/flox),named EP4 mice) was generated. TRAP staining demonstrated that there wasno significant difference in the number of TRAP⁺ osteoclasts inendplates between EP4^(f/f) and EP4^(−/−) mice of sham surgery group orLSI surgery group (FIG. 1-15A, FIG. 1-15B). Asante NaTRIUM Green 2acetoxymethyl (ANG-2 AM), a sodium indicator, was loaded into the DRGneurons to detect the real-time sodium influx. Interestingly, PGE2significantly stimulated the enhancement of the fluorescent intensity inneurons (FIG. 1-6A, left and FIG. 1-6B), indicating increased sodiuminflux. Importantly, this effect was abolished in the DRG neurons ofEP4^(−/−) mice (FIG. 1-6A, right and FIG. 1-6C). To determine themechanism by which PGE2 induces sodium influx, whether PGE2 can activatethe cyclic adenosine monophosphate (cAMP) pathway in sensory neurons wasexamined. Western blot and fluorescent staining demonstrated thatPGE2-induced cAMP production activates protein kinase A (PKA) and cAMPresponse element binding (CREB) protein, and the activation wasabrogated by PKA inhibitor or EP4^(−/−) (FIG. 1-6D-FIG. 1-6F). Further,PGE2-induced sodium influx was ablated by PKA inhibitor or smallinterfering ribonucleic acid (siRNA) for Na_(v) 1.8 (FIG. 1-6G, first tothird columns and FIG. 1-6H-FIG. 1-6J), and cAMP rescued sodium influxin EP4^(−/−) mice (FIG. 1-6G, fourth column and FIG. 1-6K). Theseresults demonstrate that PGE2 activates EP4 in sensory, neurons toinduce sodium influx of Na_(v) 1.8 through cAMP signaling withimplications for pain transduction.

To evaluate whether the PGE2/EP4 pathway in sensory fibers is associatedwith spinal pain behavior, pain behavior tests, including pressuretolerance, spontaneous activity, and von Frey analysis, were performed.The threshold of pressure tolerance in response to pressure stimulationwas lower in EP4^(−/−) mice relative to EP4^(f/f) mice after LSI surgery(FIG. 1-7A). Analysis of spontaneous activity revealed that the distancetraveled, maximum speed, mean speed, and activity time per 24 hours weresignificantly preserved in EP4^(−/−) mice relative to EP4^(f/f) f miceafter LSI surgery (FIG. 1-7B-FIG. 1-7E). Consistently, mechanicalhyperalgesia of the hind paw also was ameliorated in EP4^(−/−) micerelative to EP4^(f/f) mice after LSI surgery (FIG. 1-7F, FIG. 1-7G).

1.3.5 Decreased Osteoclast Attenuated Sensory Innervation and Pain

To evaluate whether CGRP⁺ sensory innervation in the endplates isinduced by osteoclasts, Dmp1-Cre mice were bred with osteocyte-derivedreceptor activator of nuclear factor kappa-B ligand (Rankl) floxed miceto generate Dmp1^(Cre): Rankl^(flox/flox) mice (named Rankl^(−/−) mice)to knock out Rankl specifically in DMP1⁺ osteocytes. Deficiency of Ranklin osteocytes leads to a decrease in osteoclast number and a severeosteopetrotic phenotype. Nakashima et al., 2011: Xiong et al., 2011. Thetrabecular BV/TV, Th.N, and Tb.Th increased and Tb.Sp decreasedsignificantly in RANKL^(−/−) mice relative to RANKL^(f/f) mice in μCTanalysis (FIG. 1-16A-FIG. 1-16F), indicating the osteopetrotic vertebraein Rankl-mice. The sclerosis of endplates was delayed significantly inRankl^(−/−) mice relative to their age-matched wild-type (WT)littermates (Rankl^(f/f) mice) after LSI surgery, as shown by decreasesin the porosity and trabecular separation of endplates by μCT analysis(FIG. 1-8A-FIG. 1-8C). The delayed sclerosis of endplates in Rankl^(−/−)mice was further validated by Safranin O and fast green staining, withless porous areas and significant lower endplate scores (FIG. 1-8D, FIG.1-8E). The number of TRAP⁺ osteoclasts decreased significantly inendplates at 4 and 8 weeks after LSI surgery in Rankl^(−/−) micerelative to Rankl^(f/f) mice (FIG. 1-8F, FIG. 1-8G). Notably,immunostaining showed that instability-induced CGRP⁺ sensory innervationin the endplates was inhibited in Rankl^(−/−) mice (FIG. 1-8H, FIG.1-8I). Although the density of CGRP⁺ sensory nerve fibers increasedslightly at 8 weeks after LSI surgery in Rankl^(−/−) mice, it was stillsignificantly lower than that of Rankl^(f/f) mice (FIG. 1-8H, FIG.1-8I), indicating that osteoclast activity was associated with CGRP⁺sensory innervation in the endplates. Moreover, the sprouting ofCD31⁺EMCN⁺ blood vessels in the endplates was also significantlyinhibited in Rankl^(−/−) mice relative to Rankl^(f/f) mice (FIG. 1-17A,FIG. 1-17B).

To examine whether osteoclast activity-induced sensory innervation inendplates is associated with spinal pain behavior, pain behavior tests,including pressure tolerance, spontaneous activity, and von Freyanalysis, were performed. The threshold of pressure tolerance wassignificantly lower in Rankl^(−/−) mice relative to Rankl^(f/f) miceafter LSI surgery (FIG. 1-8J). Analysis of spontaneous activity revealedthat the distance traveled, maximum speed, mean speed, and activity timeper 24 hours were significantly preserved in Rankl^(−/−) mice relativeto Rankl^(f/f) mice after LSI surgery (FIG. 8K-FIG. 8N). Consistently,mechanical hyperalgesia of the hind paw was also ameliorated inRankl^(−/−) mice relative to Rankl^(f/f) mice after LSI surgery (FIG.1-8O, FIG. 1-8P). Nerve innervation in the annulus fibrosus (AF) hasbeen implicated in back pain development. Ohtori et al., 2015. CGRP⁺sensory innervation was observed in the AF of LSI mice. There is nosignificant difference in the density of newly innervated sensory nervesbetween Rankl^(−/−) mice and Rankl^(f/f) mice (FIG. 1-18A-FIG. 1-18B).Taken together, these results indicate that sensory innervation invertebral endplates is associated with osteoclast activity and painbehavior.

1.3.6 Knockout of Netrin-1 Abrogated Sensory Innervation and Pain

Netrin-1 is an important axon guidance factor to attract nerveprotrusion. Moore et al., 2012; Serafini et al., 1996: and Hand andKolodkin, 2017. It was previously reported that osteoclasts can secretNetrin-1 to attract sensory nerve growth. Zhu et al., 2019.Immunostaining demonstrated that TRAP⁺ osteoclasts were the source ofNetrin-1 in sclerotic endplates after LSI surgery (FIG. 1-9A). The levelof Netrin-1 in lower lumbar endplates increased gradually at 4, 8, and12 weeks after LSI surgery relative to sham surgery mice, as indicatedby ELISA (FIG. 1-9B). Deleted in colorectal cancer (DCC) is identifiedas the receptor that mediates Netrin-1-induced neuronal sprouting.Forcet et al., 2002; Shu et al., 2000. Immunostaining revealed that DCCwas co-localized with CGRP⁺ sensory nerve fibers in endplates after LSIsurgery (FIG. 1-9C).

To determine whether osteoclast-derived Netrin-1 is responsible forsensory innervation, Trap-Cre mice were cross-bred with Netrin-1 floxedmice to knockout Netrin-1 in TRAP⁺ lineage cells (Trap^(Cre);Netrin-1^(flox/flox), named Netrin-1^(−/−) mice). Safranin O and fastgreen staining demonstrated that the instability-induced sclerosis ofendplates was not different in Netrin-1^(−/−) mice compared with theirage-matched littermates (herein, Netrin-1^(f/f) mice) (FIG. 1-9D), asevidenced by endplate scores (FIG. 1-9E). TRAP staining showed similarnumbers of osteoclasts in the endplates of Netrin-1^(−/−) mice andNetrin-1^(f/f) mice after LSI surgery (FIG. 1-9F, FIG. 1-9G), suggestingthat Netrin-1 is not involved in endplate sclerosis. The density ofCGRP® nociceptive nerves did not increase in Netrin-1^(−/−) mice,although the number of TRAP⁺ osteoclasts significantly increased in theendplates of Netrin-1^(−/−) mice after LSI surgery (FIG. 1-9H, 1-9I).Moreover, the sprouting of CD31⁺EMCN⁺ blood vessels in the endplates wassignificantly inhibited in Netrin-1^(−/−) mice relative toNetrin-1^(f/f) mice (FIG. 1-19A. FIG. 1-19B).

Pain behavior tests demonstrated that pressure hyperalgesia (FIG. 1-9J),loss of spontaneous activity (FIG. 1-9K-FIG. 1-9N), and mechanicalhyperalgesia of the hind paw (FIG. 1-9O, FIG. 1-9P) were significantlylower at 4 and 8 weeks after LSI surgery in Netrin-1^(−/−) mice relativeto Netrin-1^(f/f) mice. As in Rankl^(−/−) mice, CGRP⁺ sensoryinnervation in the AF did not decrease in Netrin-1^(−/−) mice (FIG.1-20A, FIG. 1-20B). Together, these results indicate thatosteoclast-derived Netrin-1 mediates sensory innervation in vertebralendplates responsible for spinal pain behavior.

1.4. Discussion

LBP is difficult to diagnose and treat because of limited knowledgeabout its source. Current treatments, including activity modification,physical therapy, and pharmaceutical agents aim to alleviate the pain,but not to modify the disease. Maher et al, 2017: Foster et al., 2018.Surgical treatment, such as disc replacement and lumbar fusion, are themost common final treatments. Efforts to understand the causes of LBPhave focused largely on sensory innervation in the degenerative IVD.Garcia-Cosamalon et al., 2010. However, IVD degeneration is frequentlyasymptomatic. Importantly, endplates undergo sclerosis during aging andbecome porous, which is clinically associated with LBP. Lotz et al.,2013; Dudli et al., 2016; and Brown et al., 1997. It has previously beenshown that aberrant mechanical loading induces sclerosis of theendplates with elevated osteoclast activity and activates excessiveTGF-01 to recruit mesenchymal stromal cells. Bian, 2016. Here, elevatedlevel of PGE2 and sensory innervation in the porous sclerotic endplateswas observed. PGE2 could activate its receptor EP4 in the newlyinnervated sensory nerves to cause spinal pain. It was recently reportedthat sensory nerves can monitor bone density changes through theconcentration of PGE2. Osteoblast-secreted PGE2 activates its receptorEP4 in the sensory nerves to tune down sympathetic tone for osteoblasticbone formation. Chen et al., 2019. The porosity of the scleroticendplates resembles low bone mineral density to promote PGE2concentration and sensory innervation causing pain. These resultsindicate that PGE2 is a key mediator of pain hypersensitivity andendplate sclerosis. These findings suggest that inhibition of endplatesclerosis to reduce sensory innervation or blocking the PGE2/EP4 pathwaycould ameliorate spinal pain behavior.

Sensory nerves and CD31⁺EMCN⁺ vessels appeared largely in the porousareas of the sclerotic endplates. Osteoclast resorption likely causesthe porosity of sclerotic endplates. Osteoclastic lineage cells secreteNetrin-1 to induce CGRP⁺ sensory nerve fiber extrusion and innervationin the space generated by osteoclast resorption. In addition, Netrin-1is suggested to be a potential pro-angiogenic factor that promotesendothelial cell migration and capillary-like tube formation. Park etal., 2004; Tu et al., 2015. It has been shown that pre-osteoclastssecrete platelet-derived growth factor-BB to induce angiogenesis coupledwith osteogenesis during bone formation. Xie et al., 2014. Thus,osteoclast activity in the sclerotic endplates is the primary cause ofsensory innervation and angiogenesis for LBP. Indeed, decreasedosteoclasts in Rankl^(−/−) mice led to significantly decreased sensoryinnervation and angiogenesis in the endplates. Moreover, the density ofsensory innervation and angiogenesis was reduced significantly byconditional knockout of Netrin-1 in TRAP⁺ osteoclastic cells. Therefore,osteoclast lineage cells instigate porosity of sclerotic endplates andspinal pain behavior.

The IVD and the endplate act in as a functional unit in the spine. Aprevious study revealed that aberrant mechanical loading inducedhypertrophy of chondrocytes and calcification of the endplates, leadingto an osteoclast-initiated remodeling sclerotic process. As a result,the expansion of mineralized endplates narrowed the intervertebral spaceand generated pathological static compression on the intervertebraldisc, leading to its degeneration. Bian, 2016. The AF has been suggestedas the main source of discogenic pain. In the physiological condition,only the outer third of the AF is innervated by sensory nerves, whereasin degenerative discs, the nerve can grow into the middle third, or eventhe inner third of the AF. Ohtori et al, 2015. In the current study,innervation of CGRP⁺ sensory fibers into the AF in WT mice after LSIsurgery was observed, but the LSI-induced newly innervated sensorynerves in the AF were not decreased in Rankl^(−/−) and Netrin-1^(−/−)mice. On the other hand, during sclerosis of the endplates, osteoclastresorption generates porous areas and abundant sensory innervation alongwith angiogenesis. Importantly, the density of CGRP⁺ sensory nervefibers in endplates and spinal pain behavior were significantlyameliorated in Rankl^(−/−) and Netrin-1^(−/−) mice after LSI surgery.These results indicate that osteoclast-induced sensory innervation inthe sclerotic endplates is the primary source of nociceptors for spinalpain.

The sign of the hind paw mechanical allodynia is considered as thesecondary indicator of spinal pain-associated behaviors. Several studiesreported that the hind paw mechanical allodynia develops in low backpain animal models as the secondary hypersensitivity. Shuang et al.,2015; Kim et al., 2011; Millecamps et al., 2015: and Kim et al., 2015.Among these works of literature, one study about the lumbar facet jointosteoarthritis-induced spinal pain excluded the local inflammation ornerve injury (with negative straight leg raising test). Kim et al.,2015. These data also show the development of hind paw mechanicalallodynia in the LSI model. One study demonstrated that the mousesciatic nerve predominantly origins from the L3 and L4 DRG by injectingretrograde labeling in the hind paw. Rigaud et al., 2008. The presentlydisclosed retrograde tracing data demonstrated that L3 DRG is also thepartial origin of sensory nerves in the endplates of L4/5 in LSI mice.In addition, the dorsal horn of spinal cord receives inputs from severalsegmental DRGs. Traub and Mendell, 1988; Kato et al., 2004. The majormonosynaptic input for the dorsal horn neurons in L4 segment is from theL4-L6 DRGs, the dorsal horn neurons in L3 segment is from the L2-L5DRGs. Pinto et al., 2008. These anatomical features might be the basisof the hind paw mechanical hypersensitivity in the LSI model.

1.5. Methods 1.5.1 Human Subjects

Human endplate samples were obtained from patients undergoing spinalfusion surgery in the Department of Spine Surgery at Xiangya Hospital(Changsha, China). Ethics committee approvals and patient consent wereobtained before harvesting human tissue samples. Detailed informationabout the patients and groups is provided in Table 1.

1.5.2 Mice

C57BL/6J (WT) male mice were purchased from Charles River Laboratories(Wilmington, Mass.). The mice were anesthetized at 2 months of age withketamine (Vetalar, Ketaset, Ketalar; 100 mg/kg, intraperitoneally) andxylazine (Rompun, Sedazine, AnaSed; 10 mg/kg, intraperitoneally). Then,the LSI model was created by resecting the L3-L5 spinous processes andthe supraspinous and interspinous ligaments to induce instability of thelumbar spine. Bian, 2016: Ariga et al., 2001; Miyamoto et al., 1991.Sham operations were performed by only detachment of the posteriorparavertebral muscles from L3-L5 on a separate group of mice. For thetime-course experiments, mice were euthanized with an overdose ofisoflurane (Forane, Baxter) inhalation at 2, 4, 8, or 12 weeks after LSIor sham surgery (10-12 per group). For the aging-induced endplatedegeneration model, 3- and 20-month-old C57BL/6J (WT) male mice werepurchased from Jackson Laboratory (10-12 per group).

The Avil-Cre mouse strain was provided by Dr. Xinzhong Dong (The JohnsHopkins School of Medicine, Baltimore, Md.). The EP4^(flox/flox) mousestrain was obtained from Dr. Brian L. Kelsall (National Institutes ofHealth, Bethesda, Md.). The Trap-Cre mouse strain was obtained from Dr.J. J. Windle (Virginia Commonwealth University, Richmond, Va.). TheNetrin-1^(flox/flox) mouse strain was provided by Dr. H. K Eltzschig(University of Texas Health Science Center at Houston, Houston. Tex.).Dnp1-Cre and Rankl^(flox/flox) mouse strains were purchased from theJackson Laboratory (Bar Harbor, Me.).

Heterozygous male Avil-Cre mice were crossed with EP4^(flox/flox) mice.The offspring were intercrossed to generate the following genotypes: WT,Avil-Cre (mice expressing (re recombinase driven by Advilin promoter).EP4^(flox/flox) (mice homozygous for the EP4 flox allele, referred to asFP4^(f/f) herein) and Avil-Cre, EP4^(flox/flox) (conditional deletion ofEP4 in Advillin lineage cells, referred to as EP4^(−/−) herein).Heterozygous Dmp1-Cre mice were crossed with Rankl^(flox/flox); theoffspring were intercrossed to generate the following genotypes: WT,Dnp1-Cre (mice expressing Cre recombinase driven by Dmp1 promoter),Rankl^(flox/flox) (mice homozygous for the Rankl flox allele, referredto as Rankl^(f/f) herein), Dmp1-Cre; Rankl^(flox/flox) (conditionaldeletion of Rankl in DMP1⁺ lineage cells, referred to as Rankl^(−/−)herein) mice. Heterozygous Trap-Cre mice were crossed withNetrin-1^(flox/flox); the offspring were intercrossed to generate thefollowing genotypes: WT, Trap-Cre (mice expressing Cre recombinasedriven by Trap promoter). Netrin-1^(flox/flox) (mice homozygous for theNetrin-1 flox allele, referred to as Netrin-1^(f/f) herein). Trap-Cre:Netrin-1^(flox/flox) (conditional deletion of Netrin-1 in TRAP⁺ lineagecells, referred to as Netrin-1^(−/−) herein) mice. The genotypes of themice were determined by PCR analyses of genomic DNA, which was extractedfrom mouse tails within the following primers: Avil-Cre: Forward:5′-CCCTGTTCACTGTGAGTAGG-3′ (SEQ ID NO: 1). Reverse:5′-GCGATCCCTGAACATGTCCATC-3′(SEQ ID NO: 2). WT:5′-AGTATCTGGTAGGTGCTTCCAG-3′(SEQ ID NO: 3): FP4 loxP allele Forward:5′-TCTGTGAAGCGAGTCCTTAGGCT-3′(SEQ ID NO: 4). Reverse:5′-CGCACTCTCTCTCTCCCAAGGAA-3′(SEQ ID NO: 5): Dmp1-Cre. Forward:5′-CCCGCAGAACCTGAAGATG-3′(SEQ ID NO: 6), Reverse:5′-GACCCGGCAAAACAGGTAG-3′(SEQ ID NO: 7), Rankl loxP allele: Forward:5′-CTGGGAGCGCAGGTTAAATA-3′(SEQ ID NO: 8), Reverse:5′-GCCAATAATTAAAATACTGCAGGAAA-3′(SEQ ID NO: 9); Trap-Cre: Forward:5′-ATATCTCACGTACTGACGGTGGG-3′(SEQ ID NO: 10), Reverse:5′-CTGTTTCACTATCCAGGTTACGG-3′(SEQ ID NO. 11); Netrin-1 loxP allele:Forward: 5′-AGGTAAAGTCTCCTACGCGG-3′(SEQ ID NO: 12). Reverse:5′-CTTCCAAACCTGAACCGCCC-3′(SEQ ID NO: 13). LSI or sham surgery wasperformed in 2-month-old male EP4^(f/f), EP4^(−/−), Rankl^(−/−),Rankl^(−/−), Netrin-1^(f/f), and Netrin-1^(−/−) mice. These mice wereeuthanized with an overdose of isoflurane inhalation at 4 or 8 weeksafter LSI or sham surgery (12 per group) All mice were maintained at theanimal facility of The Johns Hopkins University School of Medicine. Allexperimental protocols were approved by the Animal Care and UseCommittee of The Johns Hopkins University, Baltimore. Md.

1.5.4 μCT

Mice were euthanized with an overdose of isoflurane inhalation andflushed with phosphate-buffered saline (PBS) for 5 min followed by 10%buffered formalin perfusion for 5 min via the left ventricle. Then, thelower thoracic and whole lumbar spine were dissected and fixed in 10%buffered formalin for 48 h, transferred into PBS, and examined byhigh-resolution sCT (Skyscan1172). The scanner was set at a voltage of55 kVp, a current of 181 μA, and a resolution of 9.0 μm per pixel tomeasure the endplates and vertebrae. The ribs on the lower thoracicspine were included for identification of L4-L5 unit localization.Images were reconstructed and analyzed using NRecon v 1.6 and CTAn v 1.9(Skyscan US, San Jose, Calif.), respectively. Coronal images of theL4-L5 unit were used to perform 3-dimensional histomorphometric analysesof the caudal endplate. Coronal images of the L5 vertebrae were used toperform 3-dimensional histomorphometric analyses of the trabecular boneor cortical bone (anterior shell). Three-dimensional structuralparameters analyzed were total porosity and Tb.Sp for the endplates,trabecular BV/TV, Tb.N. Tb.Th, and Th. Sp for L5 vertebrae. Sixconsecutive coronal-oriented images were used for showing 3-dimensionalreconstruction of the endplates and the vertebrae using 3-dimensionalmodel visualization software. CTVol v2.0 (Skyscan US).

1.5.5 Histochemistry, Immunohistochemistry, and Histomorphometry

At the time of euthanasia, the L3-L5 lumbar spine and DRGs werecollected and fixed in 10% buffered formalin for 48 h or overnight.Then, the spine samples were decalcified in 10% or 0.5M EDTA (pi 7.4)for 14 or 5 d and embedded in paraffin or optimal cutting temperature(OCT) compound (Sakura Finetek. Torrance. Calif.). Four-μm-thickcoronal-oriented sections of the L4-L5 lumbar spine were processed forSafranin O and fast green, TRAP (Sigma-Aldrich), andimmunohistochemistry staining using a standard protocol. Thirty-μm-thickcoronal-oriented sections were prepared for sensory nerve- and bloodvessel-related immunofluorescent staining, and ten-μm-thickcoronal-oriented sections were used for other immunofluorescent stainingusing a standard protocol. The sections were incubated with primaryantibodies to mouse endomucin (1:50, sc-65495. Santa CruzBiotechnology), CD31 (1:50, ab28364, Abcam). CGRP (1:100, ab81887,Abcam), PGP9.5 (1:100, ab10404. Abcam), DCC (1:100, ab201260, Abcam),Netrin-1 (1:100, ab39370, Abcam), TRAP (1:200, ab185716, Abcam). Cox-2(1:100, ab15191. Abcam), EP4 (1:10, ab92763, Abcam), IB4 (1:100, I21411.Thermo Fisher Scientific, Waltham, Mass.), Na_(v) 1.8 (1:100, ab93616,Abeam) PGE2 (1:100, ab2318, Abeam) overnight at 4° C. Then, thecorresponding secondary antibodies were added onto the sections for 1 hwhile avoiding light. For immunohistochemistry, a horseradishperoxidase-streptavidin detection system (Dako) was subsequently used todetect the immunoactivity, followed by counterstaining with hematoxylin(Sigma-Aldrich). For immunofluorescent staining, the sections werecounterstained with 4′,6-diamidino-2-phenylindole (DAPI, Vector,H-1200). The sample images were observed and captured by a fluorescencemicroscope (Olympus BX51, DP71) or confocal microscope (Zeiss LSM 780).ImageJ (NIH) software was used for quantitative analysis. Endplatescores were calculated as described previously. Boos et al., 2002;Masuda et al., 2005.

1.5.6 Retrograde and Anterograde Tracing

2-month-old male C57BL/6J mice (Charles River) were used to perform LSIor sham surgery (6 per group). The mice were anesthetized with ketamineand xylazine at 8 weeks after surgery. For the retrograde nerve tracingexperiment, the caudal endplate of L4-L5 was adequately exposed with aventral approach. Then, 2 μL Dil (Molecular Probes; 2 mg/mL in methanol)was injected into the left part of caudal endplate of L4-L5 usingborosilicate glass capillaries after drilling a hole at left part ofendplate. The drilling holes were sealed with bone wax immediately afterinjection to prevent tracer leakage. After Dil injection, the wound wassutured, and a heating pad was used to protect mice during recovery fromanesthesia Mice were euthanized with an overdose of isofluraneinhalation, and the DRGs (T12-L6) were isolated for immunofluorescenceat 1 week after Dil injection. Ten-μm-thick frozen sections were used,and the Dil signals were inspected under 564-nm excitation using aconfocal microscope (LSM 780, Zeiss).

For the anterograde tracing experiment, 2-month-old male C57BL/6J mice(Charles River) were used to perform LSI or sham surgery and 3- and20-month-old male C57BL/6J mice (Jackson Laboratory) were prepared forthe aging-induced endplate degeneration model (6 per group). The agingmodel mice and the operated mice were anesthetized at 8 weeks aftersurgery with ketamine and xylazine. The L1 and L2 DRGs were adequatelyexposed with a dorsal approach. Then, 2 μL Dil (Molecular Probes; 2mg/mL in methanol) was injected into the DRGs using borosilicate glasscapillaries. After Dil injection, the wound was sutured, and a heatingpad was used to protect mice during recovery from anesthesia. Mice wereeuthanized with an overdose of isoflurane inhalation, and the L3-L5spine was collected for immunofluorescence at 1 week after Dilinjection. Thirty-μm-thick coronal-oriented frozen sections were used,and the Dil signals were inspected under 564-nm excitation using aconfocal microscope (LSM 780, Zeiss).

1.5.7 qRT-PCR

Total RNA was extracted from lumbar spinal endplate tissue samples usingTRIzol reagent (Invitrogen. Carlsbad, Calif.) according to themanufacturer's instructions. The purity of RNA was tested by measuringthe ratio of absorbance at 260 nm over 280 nm. For qRT-PCR, 1 μg RNA wasreverse transcribed into complementary DNA using the SuperScriptFirst-Strand Synthesis System (Invitrogen), then qRT-PCR was performedwith SYBR Green-Master Mix (Qiagen, Hilden, Germany) on a C1000 ThermalCycler (Bio-Rad Laboratories, Hercules, Calif.). Relative expression wascalculated for each gene by the 2⁻ ^(ΔΔ) ^(CT) method, withglyceraldehyde 3-phosphate dehydrogenase for normalization. Primers usedfor qRT-PCR are listed in Table 2.

TABLE 2 The primers sequence for qRT-PCR SEQ SEQ Target Forward primerID Reverse primer ID gene (5′-3′) NO. (5′-3′) NO. PGE TTTCTGCTCTGCAG 14GATTGTCTCCATGT 21 synthase CACACT CGTTGC (PGES) cox-2 CAGACAACATAAAC 15GATACACCTCTCCA 22 TGCGCCTT CCAATGACC IL-1β TTCAGGCAGGCAGT 16CGTCACACACCAGC 23 ATCACTC AGGTTAT IL-17 TCTCCACCCCAATG 17 CACACCCACCAGCA24 AAGACC TCTTCT IL-2 TTGTGCTCCTTGTC 18 CTGGGGAGTTTCAG 25 AACAGC GTTCCTTNF-α ALGAGCACAGAAAG 19 AGTAGACAGAAGAG 26 CAPG CGTGGT GAPDHAATGTGCCGTCGTG 20 AGTGTAGCCCAAGA 27 GATCTGA TGCCCTTC

1.5.8 ELISA

The concentrations of PGE2 and netrin-1 in the L3-L5 endplates weredetermined by using the PGE2 Parameter Assay Kit (KGE004B, R&D Systems)and Mouse Netrin-1 ELISA Kit (EKC37454, Biomatik, Wilmington, Del.)according to the manufacturer's instructions (3 per group),respectively.

1.5.9 DRG Neuron Culture

DRG neuron culture was processed as described previously. Saijilafu andZhou, 2012. Briefly, the dishes or coverslips for DRG neuron culturewere coated with 50 μL working solution containing 100 μg/mLPoly-D-Lysine and 10 g/mL Laminin at 37° C. To prepare the neuronculture medium, alpha minimum essential medium (α-MEM) was supplementedwith 1× penicillin-streptomycin solution (500 units of penicillin and500 μg of streptomycin, Gibco Laboratories, Gaithersburg, Md.), 5% fetalbovine serum (Gibco), 1× GlutaMAX-1 supplement (35050-061, Thermo FisherScientific), and the antimitotic reagents containing 20 μM5-fluoro-2-deoxyuridine (F0503, Sigma-Aldrich) and 20 μM uridine (U3003,Sigma-Aldrich). For the serum-free medium, the fetal bovine serum wasreplaced with the supplement B27. After euthanizing the 6- to 8-week-oldmice, the lumbar DRGs were harvested and stored in microfuge tubes withα-MEM medium placed on ice. DRG neurons were digested and dissociatedwith 1 mg/mL collagenase A solution (10103578001, Roche, Basel,Switzerland) in a 37° C. incubator for 90 min followed by 1× TrypLEExpress solution (15140-122, Thermo Fisher) at 37° C. for 20 min. Then,TrypLE Express solution was removed, and DRGs were washed gently 3 timeswith 1 mL prepared culture medium (containing 5% fetal bovine serum).Tissue was triturated by gently pipetting up and down 20-30 times in 1mL prepared culture medium. After trituration, the DRG neuron suspensionwas filtered (40-μm strainer) following non-dissociated tissuesettlement to the bottom of the microfuge and transferred to anothertube. After centrifugation (800 r.p.m for 4 min at room temperature),the cell pellet was resuspended and cultured in a precoated dish.

1.5.10 Western Blot

The primary DRG neurons were treated with 20 μM PGE2(14010, CaymanChemical, Ann Arbor, Mich.) for 30 min; 10 μM PKA inhibitor (H-89,BML-E1196, Enzo Life Sciences, Farmingdale, N.Y.) for 1 h. Western blotanalysis was conducted on the protein lysates from the cultured primaryDRG neurons. The supernatants of lysates were collected aftercentrifugation and separated by SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis) and then blotted on theNitrocellulose Blotting Membranes (Bio-Rad Laboratories). Specificantibodies were applied for incubation, and the proteins were detectedby using an enhanced chemiluminescence kit (Amersham Bioscience,RNP2108). The antibodies used for western blot were pCREB (1:1000, 9198,Cell Signaling Technology), CREB (1:000, 9197, Cell SignalingTechnology), pPKA (1:1000, 5661, Cell Signaling Technology), PKA(1:1000, 4782S. Cell Signaling Technology), and p-tubulin (1:2000,MA5-16308, Invitrogen). The original blots are provided in the SourceData file.

1.5.11 Sodium Indication

For sodium indication. ANG-2 AM (Teflabs, Austin, Tex.) was usedaccording to the manufacturer's protocol. Briefly, 1 mM stock solutionof ANG-2 AM was diluted to twice the original volume with a solution of20% Pluronic F-127 (Thermo Fisher Scientific) in DMSO. Then, the ANG-2AM/Pluronic F-127 solution was dispersed to final concentration at 5 μMANG-2 AM and 0.1% Pluronic F-127 with serum-free culture medium. Afterincubation for 1 h at 37° C., the cell loading medium was removed. Thecells were washed with serum-free and dye-free medium and prepared forsodium imaging. The sterile imaging buffer contained 5.4 mM KCL, 160 mMNaCL, 20 mM HEPES, 1.3 mM CaCL₂, 0.8 mM MgSO₄, 0.78 mM NaH₂PO₄, and 5 mMglucose (pH 7.4) The sodium imaging was monitored and captured using aconfocal microscope (Zeiss LSM 780).

In different sets of experiments, the DRG neurons were treated with 20μM PGE2 (14010, Cayman Chemical, Ann Arbor, Mich.) for 5 min. 10 μM PKAinhibitor (H-89, BML-E1196, Enzo Life Sciences, Farmingdale, N.Y.) for 1h, dibutyryl-cAMP (28945. Sigma-Aldrich) for 5 min, or siRNA oligo forNa_(v) 1.8 (GE Healthcare Dharmacon, Lafayette, Colo.). The siRNAtransfection was by using Lipofectamine RNAiMAX Transfection Reagent(Thermo Fisher Scientific) using a standard protocol.

For immunostaining, the DRG neurons were washed 3 times with PBS,followed by fixation by 4% paraformaldehyde (PFA) for 20 min at roomtemperature. The immunofluorescent staining used a standard protocol.The coverslips were incubated with primary antibodies to mouse CREB(1.100, 9197. Cell Signaling Technology), p-CREB (1:100, ab32096,Abcam), CGRP (1:100, ab81887, Abeam), PKA (1:100, 4782, Cell SignalingTechnology), and p-PKA (1:100, ab227848, Abcam) overnight at 4° C. Then,the corresponding secondary antibodies were added onto the coverslipsfor 1 h while avoiding light. The coverslips were counterstained with4′,6-diamidino-2-phenylindole (DAPI, Vector, H-1200). The sample imageswere observed and captured using a microscope (Olympus BX51, DP71).

1.5.12 Behavioral Testing

Behavioral testing was performed before surgery and weekly aftersurgery. All behavioral tests were performed by the same investigator,who was blinded to the study groups. Vocalization thresholds in responseto the force of an applied force gauge (SMALGO algometer: Bioseb.Pinellas Park, Fla.) were measured as pressure hyperalgesia. Yu et al.,2012.

A 5-mm-diameter sensor tip was directly pressed on the dorsal skin overL4-L5 (0.5 cm above the line connecting posterior iliac crest) while themice were gently restrained. The pressure force was increased at 50g/sec until an audible vocalization was made. The curve of pressureforce was recorded by using BIO-CIS software (Bioseb) to ensure theforce increased gradually. A cutoff force of 500 g was used to preventtissue trauma. Two tests were performed 15 min apart, and the mean valuewas calculated as the nociceptive threshold.

Spontaneous wheel-running activity was recorded using activity wheelsdesigned for mice (model BIO-ACTIVW-M, Bioseb). Cobos et al., 2012. Thesoftware enabled recording of activity in a cage similar to the mice'shome cage, with dimensions of 35×20×13 cm, and the wheel (diameter: 23cm, lane width: 5 cm) could be spun in both directions. The device wasconnected to an analyzer that automatically records the spontaneousactivity. The mice had ad libitum access to food and water. The distancetraveled, mean speed, maximum speed, and total active time during 2 dayswere evaluated for each mouse.

The hind paw withdrawal frequency in response to a mechanical stimuluswas determined using von Frey filaments of 0.7 mN and 3.9 mN (Stoelting,Wood Dale, Ill.). Mice were placed on a wire metal mesh grid coveredwith a clear plastic cage. Mice were allowed to acclimatize to theenvironment for 30 min before testing Von Frey filaments were applied tothe mid-plantar surface of the hind paw through the mesh floor withenough pressure to buckle the filaments. Probing was performed only whenthe mouse's paw was in contact with the floor. A trial consisted ofapplication of a von Frey filament to the hind paw 10 times at 1-secintervals. If withdraw occurred after application, it was recorded, andthe next application was performed similarly when the mouse's paw wasagain in contact with the floor. Mechanical withdrawal frequency wascalculated as the percentage of withdrawal times in response to 10applications.

Straight leg raising test was performed by stretching the hindlimb (kneejoint fully extended) and flexing the hip for 2 seconds. The number ofvocalizations in 5 leg stretch-and-lifts were recorded. Kim et al.,2015: Kroin et al., 2005. The negative result indicates that the nerveroot compression is not involved in the hyperalgesia developed after LSIsurgery.

1.5.13 Statistics

All data analyses were performed using SPSS, version 15.0, software (IBMCorp.). Data are presented as means±standard deviations. For comparisonsbetween two groups, unpaired, two-tailed Student's t-tests were used.For comparisons among multiple groups, one-way ANOVA with Bonferroni'spost hoc test was used. For all experiments. P<0.05 was considered to besignificant. All inclusion/exclusion criteria were preestablished, andno samples or animals were excluded from the analysis. No statisticalmethod was used to predetermine the sample size. The experiments wererandomized, and the investigators were blinded to allocation duringexperiments and outcome assessment. The same sample was not measuredrepeatedly.

Example 2 Parathyroid Hormone Attenuates Low Back Pain by ReducingSensory Innervation in Porous Endplates 2.1 Background

Low back pain (LBP) is a common clinical and public health problem. LBPprofoundly affects quality of life and daily physical activity,especially in the elderly population, increasing risk of frailty andloss of socioeconomic function. It is estimated that disability-adjustedlife-years (DALYs) caused by low back pain has risen from 17th in 1990to 13th in 2017 in China among all diseases. Zhou et al., 2019, and wasthe first leading cause of years lived with disability (YLD) worldwidein 2016. Global, regional, and national incidence, prevalence, and yearslived with disability for 328 diseases and injuries for 195 countries,1990-2016: a systematic analysis for the Global Burden of Disease Study2016. With an aging population and developing society, the socioeconomicburden is enormous. An estimated 149 million work days are lost everyyear in the United States alone because of LBP, Manchikanti, 2000, withtotal costs estimated to be $100-200 billion annually. Katz, 2006.

Several pathologies, like lumbar disc hemiation (LDH), scoliosis, tumorand infection, can result in low back pain. The gold standard therapyremains orthopedic surgical intervention, including: disc arthroplasty,decompression, artificial disc replacement, and spinal fusion. Wengerand Cifu, 2017; Hartvigsen et al., 2018. However, non-specific LBP(i.e., without known pathoanatomical cause) accounts for about 90% oftotal LBP cases, Koes et al., 2006, including the asymptomaticearly-stage disc degenerative disease (DDD).

Despite the high prevalence of non-specific LBP, no ideal interventionis available. Instead, education and reassurance, pharmacological andnon-pharmacological therapies have been adopted in various guidelines,but none of these therapies are targeted. In terms of pharmaceuticalefficacy and WHO analgesic ladder, the four major classes of medicationsindicated for acute LBP are paracetamol, opioids, non-steroidalanti-inflammatory drugs (NSAIDs, including COX-2 inhibitors), andskeletal muscle relaxants, while tricyclic antidepressants andbenzodiazepines are recommended for chronic cases. Maher et al., 2017.

Other minor classes, such as triptans, 2-agonists, and localanesthetics, have more specific, but limited effects on certain types ofpain (i.e., mostly acute) or related disorders. These medications areaccompanied with side effects ranging from addiction, respiratorydepression, constipation (opioids), gastrointestinal and cardiovasculardamage (NSAIDs), to a myriad of negative central nervous system (CNS)effects (anticonvulsants and antidepressants). Additionally, LBP isprone to recurrence after withdrawal. In all of these circumstances, itis believed that patients need not pain killers, but disease modifyingtherapies that abrogate or abolish pain. Due to the limitedunderstanding of non-specific LBP pathogenesis during early- andmiddle-staged DDD, however, there is no disease modifying drugs (DMDs)to prevent and treat it.

Most evidence presented thus far attributes low back pain to DDD in thelumbar region. Luoma et al., 2000. This evidence suggests its etiologyis multifactorial and affected by genetics, Battie et al., 2008; gender,Miller et al., 1988; aging, Powell et al., 1986; high mechanical stress,Videman et al., 1995; and disc dystrophy, Benneker et al., 2005.Gullbrand et al., 2015. The anatomically intact intervertebral disc(IVD), especially the nucleus pulposus (NP), is commonly considered anavascular organ except for the outer annulus fibrosus (AF) layer.Nutrients reach the IVD predominantly by diffusion through the vertebralendplate, so the NP has precarious nutrition. Rodriguez et al., 2012.Endplate has been demonstrated to be remodeled during aging, undergoinggradual ossification with elevated osteoclasts number and increasedporosity, Rodriguez et al., 2012, Jia et al., 2016; Bian et al, 2016;Bian et al., 2017; and Papdakis et al., 2011, thereby leading to discdegeneration. Gruber et al., 2005; Gruber et al., 2007. In previousstudies. Wei et al, 2015; Zhou et al., 2013, an intervertebral discdegeneration model was established in rhesus monkeys by injectingpingyangmycin, an anti-endothelial drug, into the sub-endplate region toblock the blood sinus in the endplate. These studies supported thedystrophy disorder hypothesis.

Moreover, the endplate degeneration also showed a high correlation withLBP. Jensen et al., 2008. Patients with osteoporosis are usuallyaffected with discogenic LBP, so this phenomenon was reproduced in anOVX-induced osteoporosis rhesus monkey model. The disc degeneration inthe OVX group was more severe than that of the control. Zhong et al.,2016. Endplate remodeling also was confirmed by increased calcificationand porosity, as well as decreased vascularization in the endplatesadjacent to the degenerated IVDs. These pathological featuresexacerbated degeneration of the IVDs in the animals with osteoporosis.The data suggested that the endplate becomes sclerotic from elevatedremodeling of the cartilage under normal physiological weight bearingduring aging. In addition, similar endplate changes were observed underaberrant mechanical loading in the lumbar spine instability (LSI) micemodel. Bian, 2016. Endplates become sclerotic after LSI because highlevels of TGF-β are activated by osteoclast-mediated resorption of thecartilage matrix. A more recent study revealed that sensory innervationaccompanies the osteoclast-mediated initiation of endplate sclerosis,resulting in LBP (unpublished results). Without wishing to be bound toany one particular theory, it is thought that LBP can be attenuated bypreventing endplate remodeling and sclerosis, ultimately slowing discdegeneration.

Parathyroid hormone (PTH), a systemic hormone that regulates calciumhomeostasis, plays a major role in orchestrating bone remodeling bymodulating the bone marrow microenvironment and the signaling of localfactors. Bikle et al., 2002; Canalis et al., 1989: Miyakoshi et al.,2001: Pfeilschifter et al., 1995: Wein and Kronenberg, 2018: Fan et al.,2017: Wan et al., 2008; Yu et al., 2012; and Qui et al., 2010.

Teriparatide (PTH(1-34′)) is an analogue of PTH and consists of thefirst (N-terminus) 34 amino acids. It is an FDA-approved anabolic agentto stimulate bone formation for treatment of osteoporosis. PTH(1-34′)not only improved bone density in patients with osteoporosis, but alsoattenuated LBP in various cases. For example, PTH ameliorated LBP for atleast 18 months after discontinuation. Fahrleitner-Pammer et al., 2011:Jakob et al., 2012, which suggests the pain relief resulted fromstructural remodeling of the spine components, apart from theimprovement of osteoporotic conditions. Although several clinicalstudies have reported similar phenomena, Fahrleitner-Pammer et al.,2011; Langdahl et al., 2016: and Koski et al., 2013, no biologicalmechanism has been described.

It also has been recently reported that the cilia of NP cells mediatesmechanotransduction to maintain anabolic activity in the IVD. Zheng etal., 2018. It was found that mechanical stress promotes transport of thetype 1 parathyroid hormone receptor (PTH1R) to the cilia and enhancesPTH signaling in NP cells. Daily injection of PTH effectively attenuateddisc degeneration of aged mice by direct signaling through NP cells.Given sensory innervation in the pores of endplate is associated withLBP, daily injection of PTH can increase bone formation and attenuatedisc degeneration in animals and provide relief of back pain. In thepresent example, the role of sensory innervation in vertebral endplatesand the remodeling of sclerotic endplates in the attenuation of LBPafter daily injection of PTH was investigated.

2.2 Results 2.2.1 PTH Attenuates Low Back Pain in Mice

To examine the potential effect of PTH on spinal degeneration and pain,lumbar spine instability (LSI) and aging mouse models were established.LSI or sham mice were injected with PTH and hyperalgesia tests ofpressure tolerance were performed for their low back pain (FIG. 2-1A).The value of pressure tolerance significantly decreased 8 weeks post LSIsurgery relative to sham surgery (FIG. 2-1B), indicating development oflow back pressure hyperalgesia. Interestingly, this low back pressurehyperalgesia was significantly attenuated with daily injection of PTH inLSI mice in comparison with vehicle (FIG. 2-1B). Spontaneous activitywas also measured by active wheel. The results showed that mouse activetime per 24 hours, active time, distance traveled, and mean speed ofmovement decreased significantly 8 weeks post LSI surgery relative tosham mice. Again, PTH treatment significantly increased the spontaneousactivity (FIG. 2-1C-FIG. 2 -E). In parallel, PTH effect on spine pain ofaged mice also was evaluated (FIG. 2-1F). Similarly, PTH significantlyincreased both the threshold of pressure tolerance (FIG. 2-1G) andspontaneous activity (FIG. 2-1H-FIG. 2-1J) in 18-month-old mice.Collectively, these results suggest that PTH could attenuate LBP duringspine degeneration.

2.2.2 PTH Increased the IVD Space by Decreasing the Volume and Porosityof Sclerotic Endplates

Whether PTH-reduced LBP modified spine degeneration also was examined.Previous studies have demonstrated that sclerosis of endplates decreasesIVD space and increased porosity and volume of the endplate in both LSIand aging mice. Indeed, endplates in 4-month sham-operation mice showedfew cavities detected in μCT images (FIG. 2-2A), which was furtherconfirmed with safranin O/fast green staining with red color indicatingnormal cartilage structures (FIG. 2-2B). Endplates in LSI or aged miceshowed increased porosity and volume (FIG. 2-2C-FIG. 2-2E) in μCT imagesand significant loss cartilage matrix in safranin O/fast green staining.The green-stained bone matrix around the cavities indicatesossification. Importantly. PTH treatment significantly decreased theporosity and volume of endplates (FIG. 2-2C-FIG. 2-2E). PTH alsosignificantly increased cartilage in LSI and aging mice quantitativeanalysis of safranin O/fast green staining (FIG. 2-2F). In addition, theheight and volume of IVD and endplate was measured (FIG. 2-2G).Unexpectedly. PTH increased the height and volume of IVD with a decreaseof endplate height in both LSI and aged mice (FIG. 2-2H). Theobservation indicates that PTH-reduced LBP is likely by improvement ofspine degeneration.

2.2.3 Sensory Innervation Decreased in PTH Remodeling of ScleroticEndplates

As previously reported, innervation of nerve fiber in the porousendplates or the inner 2/3 of annulus fibrosus were the sources of LBP.The change of sensory innervation in the sclerotic endplates and annulusfibrosus after iPTH was then examined. PGP9.5 is a broad marker of nervefibers. Immunostaining of intervertebral disc sections with PGP9.5showed that there is no PGP9.5 positive fibers in the sham endplatesexcept for the periphery of annulus fibrosus (FIG. 2-3A), but itsignificantly increased in endplate of LSI and aged mice relative tosham-operated mice (FIG. 2-3B). Importantly. PGP9.5 positive nervefibers were significantly reduced in the endplates of LSI and aged micewith iPTH treatment relative to vehicle (FIG. 2-3C). Although they wereless increased in the annulus fibrosus after LSI and aged mice relativeto sham-operated mice, there were no statistical differences in neitherthe vehicle group nor PTH treatment (FIG. 2-3D). This observationindicates that innervation of nerve fiber in the porous endplates, butnot annulus fibrosus, were the primary source of LBP and affected byPTH.

To determine which kinds of nerve fibers grow into the endplate, aretrograde of DIL was performed by injecting it into L4-5 endplate aspreviously reported. L1-L2 DRG sections were immuno-stained withantibodies against PGP9.5, CGRP, IB4, P2X3, PIEZO2 or NF200 respectivelyfor different sensory nerve fibers. The results showed that there wereno DIL positive neuron in sham-operated mice (FIG. 2-3E). DIL positiveneuron appeared in LSI and aging mice, but were significantly decreasedin PTH treatment groups. In addition. DIL⁺ neurons were stained withCGRP, NF2100, especially in PIZO2, but not P2X3 or IB4, which means thenerve fiber from CGRP. NF200 and PIZO2 positive neuron grows intoendplates (FIG. 2-3G). These markers also were immune-stained inendplate (FIG. 2-3E). The results also confirmed the phenomenon (FIG.2-3F). It suggested innervation porosity endplates of peptidergicnociceptive C nerve fibers and especially mechanical pain related nervefiber may be the source of pain and PTH may reduce the pain by reductionof sensory nerve fibers.

2.2.4 PTH-Induced Remodeling of Sclerotic Endplates Reduces theirPorosity

To understand the potential mechanism of PTH treatment in increase ofIVD space and reducing sensory innervation, whether PTH inducesremodeling of sclerotic endplates was examined. Trap staining andosteocalcin immunostaining revealed that osteoclasts and osteoblastswere not observed in the endplates of sham operated mice, butsignificantly increased in the endplates post LSI surgery and aged mice(FIG. 2-4A). Particularly, the osteoclasts were primarily localizedwithin the cavities of cartilage resorption in the vehicle-treated mice.Importantly, the number of osteocalcin-positive osteoblastssignificantly increased in the cavities of endplates (FIG. 2-4B, FIG.2-4G), but there were no changes of trap-positive osteoclasts inPTH-treated LSI aged mice (FIG. 2-4F), and the size of cavities weresignificantly smaller relative to the vehicle group. Moreover,CD31⁺EMCN⁺ immunofluorescence staining demonstrated that the type Hblood vessels, which is specifically associated with bone formation,were barely detectable in the endplates of sham operated mice. LSIsurgery and aged mice (FIG. 2-4C). However, PTH significantly increasedthe numbers of CD31⁺EMCN⁺ type H blood vessels in the endplates in bothLSI mice and Aging mice relative to vehicle group, indicatingPTH-induced anabolic bone formation in the cavities (FIG. 2-4H), whichwas validated by double labeling experiment (FIG. 2-4D). Golder stainingfurther demonstrated that PTH significantly increased osteoid formationat the surface of cavities in the LSI and aged mice as compared with thevehicle-treated mice (FIG. 2-3F), which is consistent with an increaseof osteoblasts in the cavities.

As the sensory nerve fibers were detected in the cavities, axonattractive factor Netrin-1, NGF played an important role in sensoryinnervation in the endplates. To examine if axon attractive andrepulsive factors play a role in the reduction of sensory nerve fibersafter iPTH, the mRNA expression of the potential factor by qPCR wasmeasured. The results showed that iPTH stimulated expression of Slit-3and Sema-3a in the endplates of LSI, but reduced the expression of NGFand inflammatory factors including PGE2 related genes although there wasno significant change in netrin-1 in the bone formation. The observationsuggested iPTH-induced decrease of porosity and increase of secretion ofaxon repulsive factors likely results in reduction of sensoryinnervation in the endplates.

2.25 IPTH Decreased Cox-2 Expression and PGE2 Levels by ReducingPorosity of Sclerotic Endplates

It has been previously shown that PGE2 secreted by osteoblastic cellsactivates PGE2 receptor 4 (EP4) in sensory nerves to maintain bonehomeostasis by modulation of sympathetic activity through the centralnervous system (Chen et al., 2019) and PGE2 levels in the bone arepositively correlated with bone density. Porous endplates resembleosteoporotic bone elevated PGE2 levels, which stimulate sensory nervesthat lead to LBP (in press). Thus, whether porosity structure ofendplates enhances mechanical loading-stimulated PGE2 secretion byfinite element analysis was examined (FIG. 2-5A). The results showedthat high-stress concentration occurred in the large cavities area ofsclerotic endplates of LSI and aging mice relative to sham-operatedmice, in which there was no high-stress concentration occurred in thewhole endplates. Stress concentrations were significantly released iniPTH-treated LSI and aged mice compared to vehicle group as porousendplates were remolded to smaller cavities (FIG. 2-5C). Therefore,whether PGE2 protein levels also changed in the endplates with iPTHtreatment was examined. The results showed that elevated PGE2 levels andCOX-2 expression were observed in ELISA (FIG. 2-5F) and immunostainingin the sclerotic endplates of LSI and aged mice relative tosham-operated mice (FIG. 2-5B). iPTH significantly reduced COX-2expression and PGE2 levels in the sclerotic endplates of LSI and agedmice relative to vehicle group (FIG. 2-5D, FIG. 2-5E). Thus,iPTH-induced remodeling of sclerotic endplates reduces PGE2 levels forLBP.

2.2.6. Knock Out PTH Receptor in Osteoblast Cell but not NucleusPulposus Cell Blunted the Effect of PTH Attenuating Spine Pain

PTH binds to the type 1 parathyroid hormone receptor (PTH1R), which isexpressed in high levels in osteoblast and osteocyte cells in bone andregulates bone homeostasis through activation of adenylate cyclase andphospholipase C. Deletion of PTH1R ensures blocking of the PTH signalingcascade. Knockout of PTH1R in osteocalcin-positive osteoblast cells ofLSI mice was induced to confirm the nerve repulsion role of PTH at theremolding of porosity endplate. Similarly to wild type mice with PTHtreatment, the total porosity and endplate volume were significantlyreduced in the PTHR f/f mice after 2 months iPTH treatment post-surgeryrelative to the vehicle treatment.

As reported in a previous study, daily injection of PTH effectivelyattenuated disc degeneration of aged mice with expressed aggrecan bydirect signaling through NP cells, which cannot only induce DRG growthcone collapse, but also inhibit DRG axonal growth. Zheng, L., et al.,2018. Knockout of PTH1R was induced in notochord cells of LSI mice toconfirm if the nerve repulsion factors from nucleus pulposus after PTHtreatment.

2.27 iPTH Attenuates Endplate Sclerosis and Disc Degeneration in AgingMonkey

To confirm the effects of iPTH in the disc degeneration, especially theendplate sclerosis, eight aging monkeys, which had similar discdegenerated grade in L1-L5 lumbar, were screened and half of them weretreated for 3 months with iPTH, the others were treated with vehicle(FIG. 2-6 ). The results of the MRI showed that the signals of disc wereincreased after iPTH 3 m comparing to pre-treatment, while the signalswere slightly decreased relative to 0 m in the vehicle group (FIG. 2-6A)And pfirrmann grade of these discs had been evaluated (FIG. 2-6E), theresults also showed iPTH resulted in lower pfirrmann grade thanpre-treatment. At the same time, there were decreased but no significantchange within 3 m in the vehicle group (FIG. 2-6F). Moreover,Quantitative analysis of T1ρ and T2 map value also exhibited similarresults, suggesting the disc degeneration had been attenuated by iPTH(FIG. 2-6B. FIG. 2-6C).

In addition, the signals change of disc after 3 monthspost-iPTH-treated-3 m also was monitored. Although the high signals ofdisc at iPTH 3 m had been reduced when stopping the injection of iPTHfor 3 m, the signals of disc at vehicle group at 6 m had become lower ascompared that at 0 m and 3 m. Further, the results of pfirrmann grade ofvehicle 6 m were significantly increased than that at 0 m. Meanwhile,the decreased values of T1ρ and T2 map within 6 m in vehicle group alsoconfirmed disc degeneration with aging, while the T1ρ and T2 map valuesincreased after iPTH treatment, especially the T2 map values continuedhigh expression in PTH group at 6 m, stopping the injection of iPTH for3 m, indicated the function of disc regeneration after iPTH.

In summary, the endplate as the main structure contributing the nutrientdiffusion from vertebrate to disc, which had been evaluated by micro-CTand tissue staining. As found in the above description of mice, largebone marrow cavities occurred in front of the endplate in aging monkey,but the porosity was significantly reduced in the iPTH-treated group.Moreover, the area of cartilage in the endplate also showed that theywere significantly increased after iPTH. Thus, these data furtherdemonstrated that iPTH can reduce sensory innervation and spine pain byremodeling of the porous endplate.

Example 3 Administration of Parathyroid Hormone Attenuates KneeOsteoarthritic Pam by Remodeling of Subchondral Bone 3.1 Overview

Osteoarthritis (OA) is a debilitating and leading prevalent jointdegeneration characterized by joint pain and disability. The currenttreatment of OA fails to attenuate OA progression and decrease jointpain effectively. Here it is shown that PTH attenuates OA pain byinhibition of nerve innervation and subchondral bone deterioration andarticular cartilage degeneration in DMM mouse model. It was found thatsensory nerve innervation for OA pain is significantly decreased inPTH-treated DMM mice compared with vehicle-treated DMM mice. Inparallel, deteriorated subchondral bone microarchitecture defined ashigher trabecular pattern factor (Tb.pf), structure model index (SMI),and increased thickness of subchondral bone plate (SBP.Th) invehicle-treated DMM control is attenuated by PTH treatment. PGE₂ insubchondral bone is increased in response to large porosity ofsubchondral bone in DMM mice. Furthermore, uncoupled subchondral boneremodeling by increased TGFβ signaling is regulated by PTH-inducedendocytosis of the PTH1R-TβRII complex. Notably, conditional knockoutthe PTH type I receptor in nestin⁺ MSCs eliminates PTH-induced improveddeteriorated subchondral bone microarchitecture, subsequent and PGE₂concentration in the subchondral bone and decreased sensory nerveinnervation for OA pain. These studies reveal that PTH attenuates OAprogression and OA pain by inhibition of subchondral bonemicroarchitecture deterioration and sensory nerve innervation.

3.2 Background

Osteoarthritis (OA) is a leading cause of disability as the most commondegenerative joint disorder, MMWR. Morbidity and mortality weeklyreport. 2009, and chronic pain is the most prominent symptom ofosteoarthitis (OA), affecting nearly 40 million people in the US. Peatet al., 2001. Pain itself is also a major risk factor for thedevelopment of future functional limitation and disability in OApatients. Lane et al., 2010. Unfortunately, OA pain treatment remainschallenging and represents a large unmet medical need. It is not clearwhat causes OA pain, and currently there is no effective way to relieveit. Available therapies (NSAIDs, steroids, visco-supplementation such asintra-articular injection of hyaluronic acid) only alleviate mild jointOA pain. Geba et al., 2002; Karlsson et al., 2002.

Relief from chronic OA pain remains an unmet medical need and stillmajor reason for seeking surgical intervention. Despite the efforts, theorigins of pain and its molecular mechanisms remain poorly understood.Significant efforts have been focused on synovium inflammation mediatedsensitization of sensory neurons. Malfait and Schnitzer, 2013. Yet, OApain can develop at very early stages without inflammation andindependently of progressive cartilage degeneration. Many asymptomaticpatients have osteoarthritic radiographic changes while other patientshave OA pain with no radiographic indications. Bedson and Croft, 2008:Dieppe and Lohmander, 2005: Hannan et al., 2000. Some patients withbilateral radiographic OA yet present with unilateral knee pain. Farless attention has been paid to subchondral bone than to synovium, andthe investigations into the mechanism how subchondral bone remodelinggenerates OA pain are lacking

The sources and mechanisms for OA pain remain unclear. Multiple tissuesincluding synovium, Kc et al., 2016, ligament, Ikeuchi et al., 2012, andmeniscus, Mapp and Walsh, 2012, were suggested to be the sites wherepain is originated. In particular, significant efforts have focused onsynovium inflammation (synovitis) and cartilage degeneration. Malfaitand Schnitzer, 2013. Intriguingly, subchondral bone marrow edema-likelesions (BMLs) are highly correlated with OA pain. Yusuf et al., 2011;Kwoh, 2013. Zoledronic acid, which inhibits osteoclasts activity,reduces the BML size and concomitantly alleviates pain. Laslett et al.,2012. Furthermore, OA patients report rapidly pain relief after removalof partial subchondral bone with deteriorated cartilage by total kneereplacement. Isaac et al., 2005; Reilly et al., 2005.

Subchondral bone remodeling is increased during OA progression. Zhen andCao, 2014. It is shown that osteoclasts initiate aberrant boneremodeling and increase the secretion of netrin-1, which triggersabnormal sensory nerve innervation in the subchondral bone and causes OApain. Subchondral bone marrow edema-like lesions (BMLs) are highlycorrelated with pain in OA patients. Kwoh, 2013; Laslett et al., 2012.

Analysis of the NIH Osteoarthritis Initiative (OAI) data also suggestedthat patients taking bisphosphonate experienced significantly reducedknee pain at year 2 and 3. Laslett et al., 2014. Bone homeostasis ismaintained by temporal-spatial activation of TGF to couple boneresorption and formation. Zhen and Cao, 2014, in which subchondral boneis maintained in a native microarchitecture with blood vessels andnerves intertwined under normal condition. However, excessive activeTGFβ in the subchondral bone induces aberrant bone remodeling at theonset of OA to promote its progression. Specifically, high levels ofactive TGFβ recruit Nestin+ MSCs and Osterix+ osteoprogenitors inclusters, leading to abnormal bone formation and angiogenesis. Zhen etal., 2013. Increased osteoclast activity is associated with angiogenesisat the onset of OA. Zhen et al., 2013; Xie et al., 2014. Since nervesand blood vessels develop together, the increased osteoclast activitylikely induces abnormal sensory nerve innervation in the subchondralbone for the OA pain.

Parathyroid hormone (PTH), an FDA-approved anabolic agent forosteoporosis, regulates bone remodeling and calcium metabolism. Qiu etal., 2010; Pfeilschifter et al., 1995; Tang et al., 200) Woolf, 2011.The parathyroid gland, the main production site of the PTH, evolved inamphibians, Mease et al., 2011, and represents the transition of aquaticto terrestrial life, Suokas et al., 2012, adapting terrestriallocomotion from aquatic vertebrates. PTH is the hormone that PTH isdemonstrated to induce cartilage regeneration for injury-induced OA,Sampson et al., 2011, Intermittent PTH stimulates subchondral bone andarticular cartilage repair in the treatment of focal osteochondraldefects. Orth et al., 2013.

The PTH is also shown to prevent the deterioration of the subchondralbone and cartilage degeneration during OA. Yan et al., 2014. It has beenshown that PTH is demonstrated to interact with locally osteopotricfactors to orchestrate with an anabolic signaling network of thecoupling of bone resorption and formation. Qiu et al., 2010;Pfeilschifter et al., 1995. TGF-β elicits its cellular response throughthe ligand-induced formation of a heteromeric complex containing TGF-βtypes I (TβRI) and II (TβRII) kinase receptors. Tang et al., 2009; Wranaet al., 1992.

Several lines of evidence have indicated that PTH and TGF-β work inconcert to exert their physiological activities in bone. PTH inducesendocytosis of PTH1R with TβRII as a complex and signaling of both PTHand TGFβ is coordinately regulated during endocytosis. Qiu et al., 2010.

This study investigates whether iPTH could attenuate pain by modifyingOA as it has positive effect on both OA subchondral bone and articularcartilage. It was found that PTH reduces OA pain and attenuatesprogression of OA by preventing subchondral bone deterioration andcartilage degeneration in OA mice. PTH reduces sensory nerve innervationin the subchondral bone and OA pain through maintaining subchondral bonemicro-architecture of sustaining of coupled bone remodeling.

3.3 Results

3.3.1 iPIH Attenuates Osteoarthritic Pain and Joint Degeneration

To investigate the effect of PTH on OA pain, PTH was administeredsubcutaneously in OA mice post DMM for two months. Pain behavior testswere performed including paw withdrawal threshold (PWT), pressureapplication measurement (PAM) and gait analysis at different timepoints(FIG. 3-1A). PWT decreased in 1- and 2-weeks post DMM and controls, andthe sham group gradually returned to the initial base level (FIG. 3-1A).iPTH significantly increased PWTs in DMM mice from 3 weeks relative toDMM vehicle group and the increase was maintained through 8 weeks.Similarly, iPTH also significantly increased PAM relative to vehiclegroup (FIG. 3-1B). Catwalk gait analysis showed that the ratio of lefthind/right hind (LH/RH) paw intensity, contact area and swing speeddecreased in vehicle DMM mice relative to sham-operated mice 8 weekspost DMM surgery, and again, iPTH significantly increased the changes inCatwalk (FIG. 3-1C and FIG. 3-1D). Fast green and Safranin O staining ofjoint sections showed that proteoglycan started loss in cartilage 2weeks post DMM in vehicle group and further aggravated at 8 weeksrelative to sham-operated group (FIG. 3-1E). iPTH reduced cartilagedegeneration in DMM group and significantly improved OsteoarthritisResearch Society International (OARSI) scores, Pritzker et al., 2006,relative to vehicle-treated DMM joint (FIG. 3-1F). Moreover, iPTH alsoreduced the percentage of MMP13⁺ and type X collagen⁺ chondrocytes inDMM mice relative to vehicle group, indicating inhibition of chondrocytedegeneration (FIG. 3-1 , FIG. 3 -G, FIG. 3 -H, FIG. 3 -I and FIG. 3 -J).Taken together, these data suggest that iPTH-reduced pain is associatedwith attenuation of cartilage degeneration in DMM OA mice.

3.3.2 iPTH Induces Regression of Sensory Nerve Fibers in SubchondralBone in OA Mice

To examine the potential mechanism of PTH-reduced OA pain, the effect ofiPTH on sensory nerve innervation in subchondral bone was examined. Theimmunostaining of subchondral bone sections revealed that calcitoningene-related peptide (CGRP)+ and substance P (SP)⁺ sensory nerve fiberssignificantly increased in vehicle-treated DMM mice relative to shamoperated mice, and iPTH ameliorated them(FIG. 3-2A, FIG. 3-2B and FIG.3-2C). Based on a newly proposed classification of sensory neurons,Usoskin et al., 2015, another 3 markers of nociceptive neurons NF200,P2X2, and PIEZO2 also were stained. The density of P2X3⁺, PIEZO2⁺, andNF200⁺ nociceptive fibers also increased in subchondral bone ofvehicle-treated DMM mice relative to sham-operated mice while iPTHtreatment significantly those fibers innervation (FIG. 3-2A. FIG. 3-20 ,FIG. 3-2E and FIG. 3-2F). In addition, there was also similar increasein PGP9.5⁺ nerve endings in DMM vehicle group, PTH treatment inducesPGP9.5⁺ nerve endings decrease to be comparable to sham group (FIG. 3-2Aand FIG. 3-2G). Furthermore, the immunostaining of CGRP⁺ sensory nervefiber and PGP 9.5⁺ nerve fiber in the joint synovium were also analyzedand showed that there were no significant differences in density ofCGRP⁺ and PGP9.5⁺ nerve between DMM PTH group and DMM vehicle group,although both of them significantly increased compared with synovium ofsham-operated DMM mice (FIG. 3-2 , FIG. 3-2H, FIG. 3-2I and FIG. 3-2J).Together, these findings suggest that intermittent PTH treatment mayameliorate OA pain by inhibition of sensory nerve innervation insubchondral bone.

3.3.3 iPTH Sustains Subchondral Bone Micro-Architecture

To examine PTH effect on subchondral bone changes in OA, PTH wasadministered daily subcutaneously in mice post DMM for 8 weeks andanalyzed the effect over time. iPTH sustained the micro-architecture oftibial subchondral bone after DMM relative to sham-operated mice and DMMvehicle mice as determined by μCT analysis (FIG. 3-3A, top). iPTHsignificantly inhibited change of subchondral bone plate (SBP),structure model index (SMI), trabecular pattern factor (Tb.Pf) and totalvolume of pore space Po.V(tot) relative to DMM vehicle group (FIG. 3-3B,FIG. 3 -C, FIG. 3 -D and FIG. 3 -E). In parallel, iPTH ameliorated theincreased expression of COX2 determined by immunohistochemical analysisand PGE2 concentration of subchondral bone determined by Elisa in DMMvehicle group (FIG. 3-3F and FIG. 3-3G). iPTH reduced abnormallocalization, as most osteoid were largely found on the bone surface,like sham-operated group, where new formed osteoid were observed insubchondral bone marrow in DMM vehicle group (FIG. 3-3H). Uncoupled boneremodeling was rescued by the PTH compared to the DMM vehicle group influorescent double labeling experiment. (FIG. 3-3I) These resultsindicated that PTH play an important role in sustaining coupledsubchondral bone remodeling in OA. Taken together, these data validatedthat PTH attenuates OA progression by decelerating subchondral bonedeterioration.

3.3.4 iPTH Attenuate Elevated Active TGF-§ Signaling by Endocytosis ofTGFβIIR

To explore the potential mechanism of PTH restoring coupled subchondralbone remodeling, the effect of PTH on osteoblast-lineage cell wasdetected. Immunostaining of nestn⁺ and osterix⁺ osteoprogenitors werelargely located on the bone surface in sham group and DMM PTH groupwhile nestin⁺ and osterix⁺ osteoprogenitors in subchondral bone marrowdramatically increased in DMM vehicle group (FIG. 3-4A, FIG. 3-4B andFIG. 3-4C). The immunostaining of pSmad2/3 revealed that the number ofpSmad2/3⁺ cell in subchondral bone was significantly decreased in DMMPTH group (FIG. 3-4D and FIG. 3-4F). The tartrate-resistant acidphosphatase-positive (TRAP) staining showed that osteoclastsignificantly increased in number in the subchondral bone post DMMrelative to sham group, and the TRAP⁺ osteoclast further increased insubchondral bone in DMM PTH group (FIG. 3-4E and FIG. 3-4G).Furthermore, Elisa of active TGFβ1 of serum revealed that iPTH induced afurther increase of the active TGFβ1 concentration relative to DMMvehicle group while a lower level of active TGFβ1 was detected insham-operated group (FIG. 3-4H). Endocytosis of PTH1R has been shown tointegrate signals of TGFβ pathways. Qiu et al., 2010.

TβRII was largely localized at mesenchymal stromal cell (MSC) membraneand the amount of cell-surface TβRII was decreased significantly afterstimulated with PTH (FIG. 3-4I). Moreover, the immunostaining ofpSmad2/3 showed that PTH decreased TGFβ1-induced phosphorylation and thenuclear accumulation of Smad2/3 relative to TGFβ1 stimulation alone(FIG. 3-4J). Collectively, high concentration of active TGFβ1 leading toaberrant subchondral bone formation, was partially prevented by PTHinduced endocytosis of TGFβIIR.

3.3.5 Delayed IPTH Attenuates Progressive Osteoarthritic Pain and JointDegeneration

To examine the effect of PTH treatment on progressive OA, a 4-weektreatment with vehicle or PTH was initiated 4 weeks post sham surgery orDMM. This delayed regimen was employed to examine the impact oftreatment in the clinical situation where the therapy is initiated aftera diagnosis of OA. Delayed regime of iPTH significantly inhibiteddecrease of PWTs and PAMs in DMM mice relative to DMM vehicle group(FIG. 3-5A and FIG. 3-5B). Catwalk gait analysis showed that iPTHinhibited decreased of the ratio of left hind/right hind (LH/RH) pawintensity, contact area and swing speed decreased in DMM vehicle mice(FIG. 3-5C) The immunostaining of subchondral bone sections revealedthat iPTH significantly ameliorated the increased density of CGRP⁺ andsubstance SP⁺ sensory nerve fibers in DMM vehicle mice (FIG. 3-5D, FIG.3-5E, and FIG. 3-5F).The degeneration of articular cartilage wasattenuated with delayed iPTH relative to DMM vehicle mice, as reflectedby SOFG staining and OARSI scores (FIG. 3-5G). Similar to immediate iPTHtreatment, delayed iPTH significantly prevented deterioration ofmicro-architecture of subchondral bone relative to DMM vehicle group(FIG. 3-5H top). The μCT analysis showed that iPTH treatment decreasedSPB.Th, SMI, Tb.Pf and tot relative to DMM vehicle group (FIG. 3-5I).The immunochemistry staining and analysis showed that iPTH reduce thenumber of COX2⁺ cells as compared to DMM vehicle group (FIG. 3 -H bottomand FIG. 3-5J). Consistently, the Elisa analysis demonstrated thatincreased PGE2 concentration of the subchondral bone in DMM vehiclegroup was significantly decreased by iPTH (FIG. 3-5J). Furthermore, iPTHsignificantly decreased the number of osterix⁺ progenitor clusters inbone marrow cavity of DMM vehicle group (FIG. 3-5K). Taken together, PTHattenuated progressive OA and subsequent sensory nerve innervation forosteoarthritic pain by preventing deterioration of subchondral bonemicrostructure.

3.3.6 Knockout of P7H1R in ASCs Inhibits PTH Induced Osteoarthritic PainRelief and Joint Degeneration Prevention

To validate PTH attenuate OA progression and OA pain through maintainingsubchondral bone microarchitecture after DMM, conditional knockout ofPTH1R was induced in nestin+ MSCs of DMM mice.Nestin-CreTMER::PTH1Rfl/fl (PTH1R^(−/−)) mice were injected withtamoxifen to delete PTH1R in the nestin⁺ MSCs, unresponsive to PTH toeliminate PTH-induced endocytosis of TGFβIIR. There were nosignificantly difference in osteoarthritic pain reflected by PWT and PAMand gait analysis between DMM PTH and vehicle PTH1R^(−/−) mice whileiPTH effectively attenuated OA pain reflected by improved PAM and PWTand gait analysis for DMM PTH1R^(fl/fl) (PTH1R^(+/+)) mice relative toDMM vehicle PTH1R^(+/+) group (FIG. 3-6A, FIG. 3-6B, and FIG. 3-6C).Moreover, CGRP⁺, SP⁺, P2X3⁺, NF200⁺ and PIZEO2⁺ sensory nerve fiber andPGP9.5⁺ nerve fibers in subchondral bone of DMM PTH PTH1R^(−/−) micewere comparable to that of DMM vehicle PTH1R^(−/−) mice (FIG. 3-6D andFIG. 3-6E). Regarding to PTH1R^(−/−) mice, iPTH significantly decreasedthe density of sensory nerve fiber relative to DMM vehicle group (FIG.3-6D and FIG. 3-6E). However, there was no statistically significantdifference in density of both CGRP⁺ and PGP9.5⁺ nerve fiber between DMMPTH and vehicle group for either PTH1R^(−/−) or PTH1R^(+/+) mice (FIG.3-6F and FIG. 3-6G).

Similarly, iPTH failed to prevent joint degeneration in DMM PTH1R^(−/−)mice. The SOFG staining showed that proteoglycan loss in articularcartilage was not prevented in the DMM PTH PTH1R^(−/−) mice (FIG. 3-7Atop and FIG. 3-7B). The micro-architecture including SMI, Tb.Pf. SBP.Thand tot were not significantly improved in DMM PTH PTH1R^(−/−) mice(FIG. 3-7A middle and FIG. 3-7C), again iPTH attenuated them in DMMPTH1R^(+/+) mice. Similarly, iPTH decreased COX2 expression determinedby immunochemistry staining and subchondral bone PGE2 concentrationdetermined by ELISA in DMM PTH1R^(+/+) mice relative DMM vehiclePTH1R^(+/+) mice, iPTH failed to decrease them in PTH1R^(−/−) mice (FIG.3-7A bottom, FIG. 3-7D and FIG. 3-7E). The number of pSmad2/3⁺ cell insubchondral bone in DMM PTH PTH1R^(−/−) mice was comparable to DMMvehicle PTH1R^(−/−) mice (FIG. 3-7F). Subsequently, iPTH failed todecrease the number of nesin⁺ osteoprogenitors and osterix⁺ in thesubchondral bone marrow failed to achieve significant difference betweenPTH and vehicle treated DMM PTH1R^(−/−) mice. Regarding to PTH1R^(+/+)mice, iPTH significantly decreased pSmad2/3⁺ cell in number and thenumber of nesin⁺ osteoprogenitors and osterix⁺ in the subchondral bonemarrow relative to DMM vehicle group (FIG. 3-7G and FIG. 3-7H). Theresults validate that PTH reduce active TGF β signaling in thesubchondral bone to prevent osteoarthritis, and further suggested thatthe role of PTH on preventing osteoarthritic pain and sustainingmicro-architecture is distinct from its role in articular cartilage.

3.4 Discussion

The current routine treatments of OA including non-steroidalanti-inflammatory drugs and analgesics have limited therapeutic effect.Hochberg et al., 2012. These drugs are palliative treatment withprogressive pathological joint changes and unsustained pain relief.Surgical joint replacement is the only alternative for end-stage of OA.Thomas et al., 2009. The only purportedly disease-modifying therapy forOA is to provide cartilage proteoglycan components via dietary or viaintra-articular injection. Zhang et al., 2008: Vangsness et al., 2009;Brzusek and Petron, 2008: Brander and Stadler, 2009.

No consensus, however, is reached on the efficacy of oral ingestion ofaggrecan sugar moieties, Zhang et al., 2008, and intra-articularinjection of hyaluronic acid have pain relief only up to 6 months.Brander and Stadler, 2009. Thus, the development of an effectivedisease-modifying treatment of the OA joint with pain relief is urgentlyneeded. In the current study, it was found that PTH could be a potentialdisease-modifying therapy of OA, considering that PTH reduce OA pain andattenuate progression of OA by preventing subchondral bone deteriorationand cartilage degeneration. The OA pain relief and prevention ofprogressive OA were due to PTH-induced maintain of subchondral bonemicro-architecture by restoration of coupled bone remodeling andprevention of nerve innervation.

In this study, no significant protection of articular cartilagedegeneration was observed when intermittent PTH was applied toNestin-CreTMER::PTH1R^(fl/fl) DMM mice compared with PTH-treated DMMPTH1R^(fl/fl) mice, suggesting that PTH-induced sustain of subchondralbone microarchitecture is critical for protection of articular cartilageduring OA. Specifically, the decreased density of CORP⁺ sensory nerve insubchondral bone in Dmp1-Rankl^(fl/fl) and Trap-Ntn^(fl/fl) is obviouslyassociated with significant pain relief reflected by Catwalk and VonFrey. Zhu et al., 2019, indicating that sensory nerve of subchondralbone might be an important origin of OA pain. OA pain of joint(activation and sensitization of nociceptive neurons) occursepisodically during movement and loading of the joint and this pain maybe evoked by specific activity such as pinch-evoked painhypersensitivity. Felson, 2009.

The primary knee hyperalgesia of OA has recently been measured bywithdrawal threshold of direct knee press using a Pressure ApplicationMeasurement (PAM) device in DMM mice. Miller et al., 2017. The secondaryhyperalgesia of knee developed after OA is also suggested to be measuredby mechanical hypersensitivity of hind paw. Zhu et al., 2019. Sing ofcentral sensitization, manifesting as pain hypersensitivity, has beendescribed, such as mechanical allodynia (pain caused by a stimulus thatdoes not normally evoke pain), reduced pain pressure thresholds, andenhanced temporal summation. Woolf, 2011: Mease et al., 2011; Suokas etal., 2012.

Various and complementary method are carried out for global measure ofOA pain in this study. The development of pain behavior involves gaitalterations due to pain during activities that shouldn't cause pain,such as pain during walking or loading. The movement-provoked painbehaviors reflected by gait analysis is effectively inhibited inPTH-treated DMM mice. The better mechanical hypersensitivity reflectedby Von Frey filament stimulation applied to operated hind paw and betterperformance of operated hind paw in PTH-treated DMM mice are consistentwith lower density of sensory nerve in subchondral bone of PTH-treatedDMM mice.

Articular cartilage and subchondral bone forms as a mechanical andbiological functional unit. Currently. OA is considered as a disease ofthe whole joint and the capacity of cross talk between articularcartilage and subchondral bone is enhanced with alteration ofsubchondral bone in OA. Zhu et al., 2019: Felson, 2009. The PTH mainlyaffected subchondral bone and articular cartilage of the joint, littleliterature established that PTH have direct effect on synovium, muscleor other soft tissues. It was commonly suggested that OA pain mainlyoriginate from synovium, ligament. Kc et al., 2016; menisci, Ashraf etal., 2011; subchondral bone, Reimann and Christensen, 1977; and muscleand joint capsule. Hirasawa et al., 2000.

The degeneration of articular cartilage, especially hyaline cartilage,unlikely itself gives rise to pain, because cartilage is normally notinnervated. Schaible, 2012. But it was reported that osteochondraljunction was innervated by sensory nerve originated from osteochondraljunction (or subchondral bone). Sun et al., 2007. It was also validatedthat there was no correlation between joint nociception and articulardamage. McDougall et al., 2009. That suggested that PTH-inducedchondro-protective and chondro-regenerative effect on articularcartilage has little relationship with pain relief during OA treatment.Additionally, PTH is also expected to promote bone resorption of old ormicro-damaged subchondral bone of OA to provide basis for new boneformation than sustain subchondral bone micro-architecture.

TrkA-positive nerve fibers were observed at innervated sites ofincipient primary ossification, coincident with NGF expression in cellsadjacent to centers of incipient ossification. Tomlinson et al., 2016.That suggest that nerve sustains bone homeostasis by locating adjacentbone surface in normal condition. Intermittent PTH spatiallyredistributes smaller blood vessels not larger vessels closer tobone-forming site for providing a favorable microenvironment for growth.Roche et al., 2014; Prisby et al., 2011.

Altogether, the PTH induced alteration of vessel suggested thatremodeling of bone vascular morphology is necessary for PTHosteoanabolic effect and its hemodynamic function. Recently, a subgroupof capillary, named type H vessel, with high level of CD31 and endomucinin the murine skeletal system was identified and found to mediatedgrowth of bone vasculature, sustain perivascular osteoprogenitors andcoupled angiogenesis to osteogenesis. Kusumbe et al., 2014.

Consistently, the type H vessel intensity significantly increased insubchondral bone of PTH treated DMM mice than that of vehicle treatedDMM mice. Osteoblast-derived VEGF were required for coupling ofangiogenesis and osteogenesis by stimulating recruitment of bloodvessels and osteoclast, to make sure blood vessel provide favorablemicroenvironment for osteogenesis. Hu and Olsen, 2016.

Type H vessel was expected to locate around bone surface in thesubchondral bone marrow of PTH treated DMM mice, which also suggestedthat osteoprogenitor cell was recruited to bone resorption bone surfaceafter PTH treated with type H vessel providing molecularmicroenvironment for coupled angiogenesis and osteogenesis. Consideringblood vessel and nerve fiber course was often alongside one another dueto sharing similar mechanism about wiring neural and vascular networks.Carmeliet and Tessier-Lavigne, 2005, it was thought that nerve fiber mayundergo similar remodeling to small vessel after PTH treatment, that mayone of reasons of PTH induced pain relief in osteoarthritis.

PGE2 secreted osteoblast that sensed lower bone volume control bonehomeostasis and promote regeneration by sensory nerve. Chen et al.,2019. The larger cavity of subchondral bone due to less loading stress(biomechanical adaptation of the bone) during OA was sense by localosteoblast, which secrets PGE₂ to sustain bone homeostasis. Bothperipheral and spinal hyperexcitability related to pathophysiologicalpain states were inhibited even reversed during development ofinflammation and established hyperexcitability by the selectivecyclooxygenase-2 (COX-2) inhibitors. Woolf and Salter, 2000;Telleria-Diaz et al., 2010.

The higher concentration of PGE2 of subchondral bone, as an inflammationfactors, may be one of main reasons of OA pain. The PTH-sustainedsubchondral bone micro-architecture with even cavity maybe associatedwith lower concentration of PGE₂, resulted in relatively mild OA pain.Additionally, intermittent PTH promoted bone formation and boosted bonemass by endocytosis of LRP6/PTH1R complex, enhancing BMP signaling. Yuet al., 2012. Subsequently, PTH-induced bone formation then inhibitedPGE2 formation due to osteoblast sensing increased bone volume.

Subchondral bone osteoblasts of osteoarthritis are resistant PTHstimulation, which could be attributed to reduced expression or alteredrecycling process of PTH1R. Hilal et al., 1998. That is consistent withreported lower PTH receptor level in OA compared to normal osteoblast.Hilal et al., 2001. Endogenous PGE₂ in subchondral bone could repressPTH-dependent response in OA osteoblasts, further contribute to abnormalbone remodeling and bone sclerosis in OA. The blunted PTH signaling dueto elevated PGE2 and IGF1, Hilal et al., 2001, signaling during OApartially explained why there only a slightly increased in BV/TV ofsubchondral bone in PTH-treated DMM mice relative to vehicle-treated DMMmice. Conversely, decrease expression of PGE2 of OA subchondral boneafter PTH treatment compared to vehicle OA mice might partially restorenormal PTH signaling, which was in part due to relatively decreasedinflammation and PTH-induced anabolic effect and sustainedmicroarchitecture of subchondral bone where PTH retards osteoarthritisprogression.

Cell senescence of osteoblasts and osteocytes have been identified withtrabecular and cortical bone and cartilage in older animals. Farr etal., 2016: Philipot et al., 2014. Given the vital role of these cells inbone remodeling and joint function, the accumulation of these senescentcells contributes to the promotion of OA. Excessive TGF-β/Smadactivation is one of the predominant pathways that acceleratedamage-induced and developmentally cellular senescence. Rapisarda etal., 2017; Lyu et al., 2018. TGF-β signaling inhibition by PTH during OAmay contribute to reduction of cellular senescence to attenuate OAprogression.

PTH was reported to provoke early osteoarthritis by induced alterationof normal subchondral bone micro-architecture. Orth et al., 2014. ThePTH-induced altered structural parameters of subchondral bone causethickening of the calcified layer, leading to osteoarthritic cartilagedegeneration. A previous study revealed that normal concentration ofactive TGF-β, as a coupling factor of bone remodeling, induces migrationof bone marrow MSCs to bone resorption site for bone formation. Tang etal., 2009.

High concentrations of active TGF-β1 signaling in the subchondral boneleads to aberrant bone remodeling, which is a key step in thepathogenesis of OA. Zhen et al., 2013. Excessive TGF β signaling ofsubchondral bone MSC in OA joint was inhibited to approach normalcondition by PTH-induced endocytosis where coupled bone remodeling waspartially restored, while the PTH-induced osteoarthritis of normalfemoral joint may be attributed to disturbance of couple bone remodelingdue to normal TGF β signaling inhibited by PTH. Furthermore, abnormallylow mineralization (becomes sclerotic although hypomineralized) ofsubchondral bone during osteoarthritis was reversed in part by PTHanabolic effect to maintain relative normal cartilage stress, Grynpas etal., 1991, while abnormally higher mineralization of normal subchondralbone induced by PTH may accelerate cartilage degeneration.

The development of osteophytes in the joint margins is a key feature ofosteoarthritis, especially in DMM model, Wright et al., 2009.Considering bone anabolic effects induced by PTH-PTH1R signaling, theremight be a potential increased incidence of osteophyte formation whenPTH was applied in the treatment of osteoarthritis. Three-dimensionalreconstructions generated from micro-CT data of all groups was collectedfor the detection of osteophyte formation. Interestingly, PTH-treatedmice did not lead to an increased incidence of osteophyte formationduring osteoarthritis compared to the vehicle-treated control group.

In the current study, the subcutaneously concentration of PTH (40μg/kg/day) is effective for the treatment of OA in DMM mice. Based onpublished literatures showing that the effective does of PTH for thetreatment of osteoarthritis ranged from subcutaneous injection (40μg/kg), Sampson et al., 2011, to intraperitoneally injection (80 μg/kg)for mice. Dutra et al., 2017, which was comparable to the concentrationhere. Notably, the dose of 40 μg/kg/day for the treatment of DMM mice inthis study was significantly higher than accepted and optimalconcentration for other species, such as guinea pig(15 μg/kg/day), Yanet al., 2014; rabbit (10 μg/kg/day), Orth et al., 2014; and rat (15μg/kg/day). Ma et al., 2017. It was anticipated that there was aspecies-associated shift for effective concentration of PTH supportingthe effect of PTH-induced OA pain relief and restoration of coupled boneremodeling then the further attenuation of OA progression.

3.5 Methods 3.5.1 Mice

C57BL6J mice (WT mice. Stock number: 000664) were purchased from JacksonLaboratory, 10-weeks old male mice were anesthetized with xylazine andketamine and then transected meniscotibial ligament that connectslateral side of medial meniscus with intercondylar eminence of tibia toinduce mechanical instability associated osteoarthritis on the leftknee, Sham operations of DMM were done on independent mice. Forimmediate treatment group, beginning 3 days after surgery, PTH (40μg/kg/day) or the equivalent volume of vehicle (PBS) was injectedsubcutaneously daily until sacrifice. For the time-course experiment,operated mice were euthanized at 2, 4, 8, and 12 weeks postoperatively(n=8 per treatment group). Regarding to delayed treatment group, PTH (40μg/kg/day) or the equivalent volume of vehicle (PBS) was injectedsubcutaneously daily from 4 weeks to 8 weeks after surgery, then thesemice were sacrificed (n=8 per treatment group).

Nestin-creERT2 mice (Stock number: 016261) and R26R-EYFP (Stocknumber:006148) were purchased from Jackson Laboratory. Mice with floxedPTH1R (PTH1R^(flox/flox)) were obtained from the lab of Dr. HenryKronenberg. Kobayashi et al., 2002.

The genotype of transgenic mice was determined by PCR analyses ofgenomic DNA isolated from mouse tails. The floxed PTH1R allele wasidentified with primers lox1F (5′-TGGACGCAGACGATGTCTTTACCA-3′ (SEQ IDNO: 28)) and lox1R (5′-ACATGGCCATGCCTGGGTCTGAGA-3′ (SEQ ID NO: 29)). Thegenotyping for the creERT2 transgene was performed by PCR with theprimers Cre F (5′-ACCAGAGACGGAAATCCATCGCTC-3′ (SEQ ID NO: 30)) and Cre R(5′-TGCCACGACCAAGTGACAGCAATG-3′ (SEQ ID NO: 31)). Nestin-creERT2:: PTH1R flox/flox mice were generated by crossing Nestin-creERT2 mice withPTH1R flox/flox mice. Then these mice were backcross with PTH1Rflox/flox mice to generate Nestin-creERT2::PTH1R flox/flox and PTH 1Rflox/flox mice. DMM or sham operations were performed on 10-week oldNestin-creERT2::PTH1R flox/flox and PTH1R flox/flox male mice. Threedays before surgery, the mice were treated with either 100 mg/kg bodyweight of tamoxifen or vehicle three times per week for 4 and 8 weeks.Additionally, the mice (n=8 per treatment group) were treated witheither PTH (40 μg/kg/day) or the equivalent volume of vehicle (PBS)subcutaneously daily for 4 and 8 weeks, 3 days after surgery.

3.5.2. Cell Culture

Bone marrow stromal cell was obtained from 8-weeks old WT mice asdescribed by Soleimani and Nadri, 2009. Cells (Passage 3-5) weremaintained in Iscove's modified Dulbecco's medium (Invitrogen)supplemented with 10% fetal calf serum (Atlanta Biologicals), 10% horseserum (Thermo Scientific), and 1% penicillin-streptomycin (Mediatech).MSCs were cultured in 6 well plates at a density of 1.8×10⁵ cells perwell, then MSC were starved for 6 h before treatment. Human PTH (1-34)and PTH (7-34) was purchased from Bachem, Inc. (Torrance, Calif.), 100nM of or PTH(7-34), or 2 ng of TGF-β1, was used for cell treatment asindicated.

3.5.3. ELISA

The concentration of active TGF-β1 in the serum was determined using theTGF-β1 ELISA Development kit (human: R&D Systems. DB100B; mouse: R&DSystems, MB100B) and following the manufacturer's instructions.

3.5.4. Histochemistry, Immunohistochemistry and Histomorphometry

After mice were killed by carbon dioxide (CO₂) inhalation and perfusedby phosphate buffer saline (PBS) and fixed by 10% buffered formalin viathe left ventricle, the left knee joints were resected and fixed in 10%buffered formalin for 24 hours, decalcified in 0.5M ethylenediaminetetraacetic acid (pH 7.4) for 21 days, and embedded in paraffin orOptimal Cutting Temperature Compound (O.C.T. compound, VWR, 25608-930).The blocks were sectioned at 4 μm intervals using a Paraffin Microtome(for paraffin blocks) or 30 μm intervals using a using a Microm cryostat(for frozen blocks). Four-μm sagittal oriented sections of the operatedknee joint medial compartment for hematoxylin and eosin (H&E) stainingand safranin o (Sigma-Aldrich, S2255) and fast green (Sigma-Aldrich,F7252) staining. Tartrate resistant acid phosphatase staining wasperformed using the manufacturer's protocol (Sigma-Aldrich, 387A-1KT),followed by counterstaining with Methyl Green (Sigma-Aldrich, M884).Immunostaining including immunohistochemistry and immunofluorescencewere performed using standard protocol. The tissue sections wereincubated with primary antibodies to mouse pSmad2/3 (ThermoFisherScientific, 444-244G, 1:100), mouse osterix (Abcam, ab22552, 1:200),mouse CD31 (Abcam, ab28364, 1:100), mouse endomucin (Santa Cruz, V.7C7,1:50), mouse osteocalcin (Abcam, ab93876, 1:200), mouse nestin (AyesLabs, Inc., 1:300, lot NES0407), mouse CGRP 0, mouse Substance P ( ),and mouse PGP9.5 ( ) overnight at 4° C. in a humidifier chamber. Thesections were washed three times with Tris-buffered saline. Forimmunohistochemical staining, the slides were incubated with secondaryantibodies in blocking solution at room temperature for 1 hours, andsubsequently Chromogenic Substrates (Dako, K3468) was used to detect theimmunoactivity, followed by counterstaining with hematoxylin(Sigma-Aldrich, H9627). For immunofluorescence staining, slides wereincubated with secondary antibodies conjugated with fluorescence at roomtemperature for 1 hour, while avoiding light and mounted on slides withProlong Gold Mounting Reagent with DAPI (Life Technologies, P36935).Isotype-matched controls, such as polyclonal rabbit IgG (R&D Systems,AB-105-C) and monoclonal rat IgG2A (R&D Systems, 54447), and polyclonalgoat IgG (R&D Systems, AB-108-C) were used as negative controls undersame condition and concentration. A Zeiss 780 confocal microscope or anOlympus DP72 microscope (Microscope Camera, Olympus, Tokyo, Japan) wasused for image capture. Quantitative histomorphometric analysis wasconducted in a blinded fashion with Image-Pro Plus Software version 6.0(Media Cybernetics Inc., Rockville, Md.). The numbers of positivestained cells in five random visual fields of five sequential sectionsper mouse in each group were counted and normalized to the number permillimeter of adjacent bone surface (for TRAP staining quantification)or per square millimeter of bone marrow. For type H vessel and nervequantification, the percentage area of positive staining was calculatedby measuring the positive area and normalized to that of sham mice per(set to 1) in each group. Quantifications were performed using ImageJ1.48u4 software.

3.5.5 Micro-Computed Tomography (μCT)

The left knee joint was dissected from mice free of soft tissue andfixed overnight in 10% formalin, and then analyzed by high resolutionμCT (Skyscan 1172). The scanner was set at a voltage of 65 kV and acurrent of 153 μA and a resolution of 9 μm per pixel. The images werereconstructed, analyzed for HO bone volume, and visualized by NReconv1.6, CTAn v 1.9, and CTVol v2.0, respectively. Cross-sectional imagesof the tibiae subchondral bone. The region of interest was defined asthe whole medial compartment of subchondral bone and cross-sectionalsagittal image of the tibiae subchondral bone were used forthree-dimensional histomorphometric analysis. A total of six consecutiveimage from medial tibial plateau were used for 3D reconstruction.Cross-sectional sigittal images of the tibiae subchondral bone were usedto perform three-dimensional histomorphometric analysis. The followingthree-dimensional structural parameters were analyzed in this study:BV/TV: trabecular bone volume per tissue volume, SMI: structure modelindex. Tb.pf: trabecular pattern factor. SBP: subchondral bonethickness, and Tb.Th: trabecular thickness.

3.5.6 Gait Analysis

Detail automated analysis of gait was performed on walking mice using a“CATWALK” system (Noldus) pre-surgery, 2, 4, 6 and 8 weeks post-surgery.All experiments were performed at the same period of the day (12:00PM-4:00 PM) and analyzed as previously reported method. Hamers et al.,2006; Hamers et al., 2001.

The recording was carried in a completely dark room with exception ofthe light from the computer screen. Briefly, mice were trained to crossthe Catwalk walkway daily for 7 days before DMM or sham operation.During the test, each mouse was placed individually in the Catwalkwalkway and allowed to walk freely and traverse form one side to theother side of the walkway glass plate. Light from an encased fluorescentlamp was emitted inside the glass plate and completely internallyreflected. When the mouse paws contacted the glass plate, light wasreflected down and the illuminated contact area was recorded with ahigh-speed color video camera positioned underneath the glass plateconnected to a computer running Catwalk software v9.1 (Noldus).Comparison was made between the ipsilateral (left) and the contralateral(right) hind paw in each run of each animal at each time point. In thepresent study, the following parameters were analyzed: contact area;intensity and swing speed.

3.5.7 Von Frey

The 50% paw withdrawal threshold was measured by Von Frey hairalgesiometry. Mice was habituated to elevated Plexiglas chambers andwire mesh flooring prior to assessments of allodynia. Then, ipsilateralhind paw mechano-sensitivity was measured by a modification of the Dixonup-down method. Dixon, 1980. Allodynia were evaluated by application ofvon Frey hair in ascending order of known bending force (force range:0.07 g, 0.4 g, 0.6 g, 1 g, 1.4 g, 2 g, 4 g, or 6 g). The von Frey hairwas applied perpendicular to the plantar surface of the hind paw(avoiding the toe pads) for 2-3 s, once a withdrawal response wasoccurred the paw was re-tested starting with the next descending vonFrey hair until no response occurred. Four more measurements were madeafter the first difference were observed. The 50% PWT was determined bythe following formula: 50% PWT=10^(Xf+kδ/)10000, where Xf is the exactvalue of the final test of von Frey hair, K is the tabular value for thepattern of the last 6 positive/negative responses, and S is the meandifference (in log units) between stimuli. The threshold force requiredto elicit withdrawal of the paw (median 50% withdrawal) was determinedtwice on each hind paw (and averaged) on each testing day, withsequential measurements separated by at least 5 min.

3.5.8. Statistics

All data were analysis were performed using SPSS 22.0 analysis software(SPSS Inc). The data are presented as the mean±standard deviation (SD).Unpaired two-tailed Student's t-test were used for comparison betweentwo groups and one-way analysis of variance (ANOVA) followed by wereused to determined significant difference between multiple groups. Thelevel of significance as set at p<0.05. All inclusion/exclusion criteriawere pre-established, and no samples or animals were excluded from theanalysis. No statistical method was used to predetermine the samplesize. The experiments were randomized. The investigators were notblinded to allocation during experiments and outcome assessment.

REFERENCES

All publications, patent applications, patents, and other referencesmentioned in the specification are indicative of the level of thoseskilled in the art to which the presently disclosed subject matterpertains. All publications, patent applications, patents, and otherreferences are herein incorporated by reference to the same extent as ifeach individual publication, patent application, patent, and otherreference was specifically and individually indicated to be incorporatedby reference. It will be understood that, although a number of patentapplications, patents, and other references are referred to herein, suchreference does not constitute an admission that any of these documentsforms part of the common general knowledge in the art.

-   Ariga, K. et al. The relationship between apoptosis of endplate    chondrocytes and aging and degeneration of the intervertebral disc.    Spine 26, 2414-2420 (2001).-   Ashraf S, Wibberley H, Mapp P I, Hill R, Wilson D, Walsh D A.    Increased vascular penetration and nerve growth in the meniscus: a    potential source of pain in osteoarthritis. Annals of the rheumatic    diseases, 2011:70(3):523-529.-   Battie, M. C., et al., Genetic and environmental effects on disc    degeneration by phenotype and spinal level: a multivariate twin    study. Spine (Phila Pa. 1976), 2008, 33(25): p. 2801-8.-   Bedson J, Croft P R. The discordance between clinical and    radiographic knee osteoarthritis: A systematic search and summary of    the literature. Bmc Musculoskeletal Disorders, 2008; 9.-   Benneker, L. M., et al., 2004 Young Investigator Award Winner:    vertebral endplate marrow contact channel occlusions and    intervertebral disc degeneration. Spine (Phila Pa. 1976), 2005,    30(2): p. 167-73.-   Bian, Q. et al. Excessive Activation of TGF beta by Spinal    Instability Causes Vertebral Endplate Sclerosis. Scientific    Reports 6. doi:ARTN 2709310.1038/srep27093 (2016).-   Bian, Q. et al. Mechanosignaling activation of TGFbeta maintains    intervertebral disc homeostasis. Bone Res 5, 17008.    doi:10.1038/boneres.2017.8 (2017).-   Bikle, D. D., et al., Insulin-like growth factor I is required for    the anabolic actions of parathyroid hormone on mouse bone. J Bone    Miner Res, 2002, 17(9): p. 1570-8.-   Boos, N. et al. Classification of age-related changes in lumbar    intervertebral discs: 2002 Volvo Award in basic science. Spine 27,    2631-2644 (2002).-   Borenstein, D. G. et al. The value of magnetic resonance imaging of    the lumbar spine to predict low-back pain in asymptomatic subjects—A    seven-year follow-up study. Journal of Bone and Joint    Surgery-American Volume 83a, 1306-1311, doi:Doi    10.2106/00004623-200109000-00002 (2001).-   Brander V A, Stadler T S. Functional improvement with hylan G-F 20    in patients with knee osteoarthritis. The Physician and sports    medicine. 2009; 37(3):38-48.-   Brown, M. F. et al. Sensory and sympathetic innervation of the    vertebral endplate in patients with degenerative disc disease. J    Bone Joint Surg Br 79, 147-153 (1997).-   Brzusek D, Petron D. Treating knee osteoarthritis with    intra-articular hyaluronans. Current medical research and opinion.    2008; 24(12):3307-3322.-   Cai, G. Q. et al. Effect of Zoledronic Acid and Denosumab in    Patients With Low Back Pain and Modic Change: A Proof-of-Principle    Trial. Journal of Bone and Mineral Research 33, 773-782,    doi:10.1002/jbmr.3376 (2018).-   Canalis, E., et al., Insulin-like growth factor I mediates selective    anabolic effects of parathyroid hormone in bone cultures. J Clin    Invest, 1989, 83(1): p. 60-5.-   Carmeliet P, Tessier-Lavigne M. Common mechanisms of nerve and blood    vessel wiring. Nature. 2005:436(7048):193-200.-   Chen H. Hu B, Lv X, et al. Prostaglandin E2 mediates sensory nerve    regulation of bone homeostasis. Nature Communications. 2019;    10(1):181.-   Clark, P. et al. MF498    [N-{[4-(5,9-Diethoxy-6-oxo-6,8-dihydro-7H-pyrrolo[3,4-g]quinolin-7-yl)-3-methylbenzyl]sulfonyl}-2-(2-methoxyphenyl)acetamide],    a selective E prostanoid receptor 4 antagonist, relieves joint    inflammation and pain in rodent models of rheumatoid and    osteoarthritis. The Journal of pharmacology and experimental    therapeutics 325, 425-434 (2008).-   Cobos, E. J. et al. Inflammation-induced decrease in voluntary wheel    running in mice: a nonreflexive test for evaluating inflammatory    pain and analgesia. Pain 153, 876-884 (2012).-   Coggeshall, R. E., Tate, S. & Carlton, S. M. Differential expression    of tetrodotoxin-resistant sodium channels Na_(v)1.8 and Na_(v)1.9 in    normal and inflamed rats. Neuroscience letters 355, 45-48 (2004).-   Dieppe P A, Lohmander L S. Pathogenesis and management of pain in    osteoarthritis. Lancet. 2005:365(9463):965-973.-   Disease, G. B. D., 1. Injury. and C. Prevalence, Global, regional,    and national incidence, prevalence, and years lived with disability    for 328 diseases and injuries for 195 countries, 1990-2016: a    systematic analysis for the Global Burden of Disease Study 2016.    Lancet, 2017, 390(10100): p. 1211-1259.-   Dixon W J. Efficient Analysis of Experimental Observations. 1980;    20(1):441-462.-   Dudli, S., Fields, A. J., Samartzis, D., Karppinen, J. & Lotz, J. C.    Pathobiology of Modic changes. European spine journal: official    publication of the European Spine Society, the European Spinal    Deformity Society. and the European Section of the Cervical Spine    Research Society 25, 3723-3734 (2016).-   Dutra E H, O'Brien M H, Gutierrez T, Lima A, Nanda R, Yadav S. PTH    [1-34]-induced alterations predispose the mandibular condylar    cartilage to mineralization. 2017; 20(S1):162-166.-   England, S., Bevan, S. & Docherty, R. J. PGE2 modulates the    tetrodotoxin-resistant sodium current in neonatal rat dorsal root    ganglion neurones via the cyclic AMP-protein kinase A cascade. J    Physiol 495 (Pt 2), 429-440 (1996).-   Fahrleitner-Pammer, A., et al., Fracture rate and back pain during    and after discontinuation of teriparatide: 36-month data from the    European Forsteo Observational Study (EFOS). Osteoporos Int, 2011,    22(10): p. 2709-19.-   Fan, Y., et al., Parathyroid Hormone Directs Bone Marrow Mesenchymal    Cell Fate. Cell Metab, 2017, 25(3): p. 661-672.-   Farr J N, Fraser D G, Wang H. et al. Identification of Senescent    Cells in the Bone Microenvironment. 2016:31(11).1920-1929.-   Felson DTJAR, Therapy. Developments in the clinical understanding of    osteoarthritis. 2009; 11(1):203.-   Fields, A. J., Liebenberg, E. C. & Lotz, J. C. Innervation of    pathologies in the lumbar vertebral end plate and intervertebral    disc. Spine Journal 14, 513-521, doi:10.1016/j.spinee.2013.06.075    (2014).-   Forcet, C. et al. Netrin-1-mediated axon outgrowth requires deleted    in colorectal cancer-dependent MAPK activation. Nature 417, 443-447    (2002).-   Foster, N. E. et al. Prevention and treatment of low back pain:    evidence, challenges, and promising directions. Lancet 391,    2368-2383, doi:10.1016/S0140-6736(18)30489-6 (2018).-   Garcia-Cosamalon, J. et al. Intervertebral disc, sensory nerves and    neurotrophins: who is who in discogenic pain? Journal of anatomy    217, 1-15 (2010).-   Geba G P. Weaver A L, Polis A B, Dixon M E, Schnitzer T J, Grp V.    Efficacy of rofecoxib, celecoxib, and acetaminophen in    osteoarthritis of the knee—A randomized trial. Jama-Journal of the    American Medical Association. 2002:287(1):64-71.-   Global, regional, and national incidence, prevalence, and years    lived with disability for 310 diseases and injuries, 1990-2015: a    systematic analysis for the Global Burden of Disease Study 2015.    Lancet 388, 1545-1602, doi:10.1016/S0140-673(416)31678-6 (2016).-   Gruber, H. E., et al., Vertebral endplate and disc changes in the    aging sand rat lumbar spine: cross-sectional analyses of a large    male and female population. Spine (Phila Pa. 1976), 2007, 32(23): p.    2529-36.-   Grynpas M D, Alpert B, Katz I, Lieberman I, Pritzker K P.    Subchondral bone in osteoarthitis. Calcified tissue international.    1991; 49(1):20-26.-   Gullbrand, S. E., et al., ISSLS Prize Winner Dynamic Loading-Induced    Convective Transport Enhances Intervertebral Disc Nutrition. Spine    (Phila Pa. 1976), 2015, 40(15): p. 1158-64.-   Hamers D F P T, Koopmans G C, Joosten E A J. CatWalk-Assisted Gait    Analysis in the Assessment of Spinal Cord Injury. 2006;    23(3-4):537-548.-   Hamers F P, Lankhorst A J, van Laar T J, Veldhuis W B, Gispen W H.    Automated quantitative gait analysis during overground locomotion m    the rat: its application to spinal cord contusion and transection    injuries. Journal of neurotrauma. 2001; 18(2):187-201.-   Hancock, M. J. et al. Systematic review of tests to identify the    disc, SIJ or facet joint as the source of low back pain. Eur Spine J    16, 1539-1550, doi:10.1007/s00586-007-0391-1 (2007).-   Hand, R. A. & Kolodkin, A. L. Netrin-Mediated Axon Guidance to the    CNS Midline Revisited. Neuron 94, 691-693 (2017).-   Hannan M T. Felson D T, Pincus T. Analysis of the discordance    between radiographic changes and knee pain in osteoarthritis of the    knee. Journal of Rheumatology. 2000; 27(6):1513-1517.-   Hartvigsen, J. et al. What low back pain is and why we need to pay    attention. Lancet (London. England) 391, 2356-2367 (2018).-   Hartvigsen, J., Christensen, K. & Frederiksen, H. Back pain remains    a common symptom in old age, a population-based study of 4486 Danish    twins aged 70-102. Eur Spine J 12, 528-534,    doi:10.1007/s00586-003-0542-y (2003).-   Hilal G, Martel-Pelletier J. Pelletier J P, Ranger P, Lajeunesse D.    Osteoblast-like cells from human subchondral osteoarthritic bone    demonstrate an altered phenotype in vitro: possible role in    subchondral bone sclerosis. Arthritis and rheumatism. 1998;    41(5):891-899.-   Hilal G, Massicotte F, Martel-Pelletier J, Fernandes J C, Pelletier    J P, Lajeunesse D. Endogenous prostaglandin E2 and insulin-like    growth factor 1 can modulate the levels of parathyroid hormone    receptor in human osteoarthritic osteoblasts. Journal of bone and    mineral research:the official journal of the American Society for    Bone and Mineral Research. 2001:16(4) 713-721.-   Hirasawa Y, Okajima S. Ohta M, Tokioka T. Nerve distribution to the    human knee joint: anatomical and immunohistochemical study.    International orthopaedics. 2000, 24(1):1-4.-   Hochberg M C, Altman R D, April K T, et al. American College of    Rheumatology 2012 recommendations for the use of nonpharmacologic    and pharmacologic therapies in osteoarthritis of the hand, hip, and    knee. Arthritis care & research. 2012:64(4):465-474.-   Hu K. Olsen B R. Osteoblast-derived VEGF regulates osteoblast    differentiation and bone formation during bone repair. The Journal    of clinical investigation. 2016:126(2):509-526.-   Hurri, H. & Karppinen, J. Discogenic pain. Pain 112, 225-228,    doi:10.10161j.pain.2004.08.016 (2004).-   Ikeuchi M. Wang Q, Izumi M, Tani T. Nociceptive sensory innervation    of the posterior cruciate ligament in osteoarthritic knees. Archives    of Orthopaedic and Trauma Surgery. 2012; 132(6)891-895.-   Isaac D, Falode T, Liu P, I′Anson H, Dillow K, Gill P. Accelerated    rehabilitation after total knee replacement. Knee. 2005;    12(5):346-350.-   Jakob, F., et al., Effects of teriparatide in postmenopausal women    with osteoporosis pre-treated with bisphosphonates: 36-month results    from the European Forsteo Observational Study. Eur J Endocrinol,    2012, 166(1): p. 87-97.-   Jarvinen, J. et al. Association between changes in lumbar Modic    changes and low back symptoms over a two-year period. BMC    Musculoskelet Disord 16, 98 (2015).-   Jarvis, M. F. et al. A-803467, a potent and selective Nav1.8 sodium    channel blocker, attenuates neuropathic and inflammatory pain in the    rat. Proceedings of the National Academy of Sciences of the United    States of America 104, 8520-8525 (2007).-   Jensen, O. K. Nielsen, C. V., Sorensen, J. S. &    Stengaard-Pedersen, K. Type I Modic changes was a significant risk    factor for 1-year outcome in sick-listed low back pain patients: a    nested cohort study using magnetic resonance imaging of the lumbar    spine. Spine J 14, 2568-2581 (2014).-   Jensen, T. S., Karppinen, J., Sorensen, J. S., Niinimaki, J. &    Leboeuf-Yde, C. Vertebral endplate signal changes (Modic change): a    systematic literature review of prevalence and association with    non-specific low back pain. European Spine Journal 17, 1407-1422,    doi:10.1007/s00586-008-0770-2 (2008).-   Jia, H., et al., Oestrogen and parathyroid hormone alleviate lumbar    intervertebral disc degeneration in ovariectomized rats and enhance    Wnt/beta-catenin pathway activity. Sci Rep, 2016. 6: p. 27521.-   Karlsson J. Sjogren L S, Lohmander L S. Comparison of two hyaluronan    drugs and placebo in patients with knee osteoarthritis. A    controlled, randomized, double-blind, parallel-design multicentre    study. Rheumatology. 2002; 41(11):1240-1248.-   Kato, G. et al. Electrophysiological mapping of the nociceptive    inputs to the substantia gelatinosa in rat horizontal spinal cord    slices. J Physiol 560, 303-315, doi:10.1113/jphysiol.2004.068700    (2004).-   Katz, J. N., Lumbar disc disorders and low-back pain: socioeconomic    factors and consequences. J Bone Joint Surg Am. 2006. 88 Suppl 2: p.    21-4.-   Kc R, Li X, Kroin J S, et al. PKCdelta null mutations in a mouse    model of osteoarthritis alter osteoarthritic pain independently of    joint pathology by augmenting NGF/TrkA-induced axonal outgrowth.    Annals of the rheumatic diseases. 2016; 75(12):2133-2141.-   Kim, J.-S. et al. Development of an Experimental Animal Model for    Lower Back Pain by Percutaneous Injury-Induced Lumbar Facet Joint    Osteoarthritis. J Cell Physiol 230, 2837-2847 (2015).-   Kim, J.-S. et al. The rat intervertebral disk degeneration pain    model: relationships between biological and structural alterations    and pain. Arthritis Res Ther 13, R165 (2011).-   Kirkby Shaw, K., Rausch-Derra, L. C. & Rhodes, L. Grapiprant: an EP4    prostaglandin receptor antagonist and novel therapy for pain and    inflammation. Veterinary medicine and science 2, 3-9 (2016).-   Kobayashi T, Chung U I, Schipani E, et al. PTHrP and Indian hedgehog    control differentiation of growth plate chondrocytes at multiple    steps. Development (Cambridge, England). 2002:129(12):2977-2986.-   Koes, B. W., M. W. van Tulder, and S. Thomas, Diagnosis and    treatment of low back pain. BMJ, 2006. 332(7555): p. 1430-4.-   Koivisto, K. et al. Efficacy of zoledronic acid for chronic low back    pain associated with Modic changes in magnetic resonance imaging.    BMC Musculoskelet Disord 15, doi:Artn (410.1186/1471-2474-15-64    (2014).-   Koski, A. M., et al., The effectiveness of teriparatide in the    clinical practice—attenuation of the bone mineral density outcome by    increasing age and bisphosphonate pretreatment. Ann Med, 2013.    45(3): p. 230-5.-   Krismer, M., van Tulder, M., Low Back Pain Group of the B. & Joint    Health Strategies for Europe, P. Strategies for prevention and    management of musculoskeletal conditions. Low back pain    (non-specific). Best Pract Res Clin Rheumatol 21, 77-91,    doi:10.1016/j.berh.2006.08.004 (2007).-   Kroin, J. S., Buvanendran, A., Cochran, E. & Tuman, K. J.    Characterization of pain and pharmacologic responses in an animal    model of lumbar adhesive arachnoiditis. Spine 30, 1828-1831 (2005).-   Kusumbe A P, Ramasany S K, Adams R H. Coupling of angiogenesis and    osteogenesis by a specific vessel subtype in bone. Nature. 2014;    507(7492):323-328.-   Kwoh C K. OSTEOARTHRITIS Clinical relevance of bone marrow lesions    in OA. Nature Reviews Rheumatology. 2013; 9(0):7-8.-   Lane N E, Schnitzer T J, Birbara C A, et al. Tanezumab for the    Treatment of Pain from Osteoarthritis of the Knee. New England    Journal of Medicine. 2010; 363(16):1521-1531.-   Langdahl, B. L., et al., Fracture Rate, Quality of Life and Back    Pain in Patients with Osteoporosis Treated with Teriparatide:    24-Month Results from the Extended Forsteo Observational Study    (ExFOS). Calcif Tissue Int, 2016. 99(3): p. 259-71.-   Laslett L L, Dore D A, Quinn S J, et al. Zoledronic acid reduces    knee pain and bone marrow lesions over 1 year: a randomised    controlled trial. Annals of the Rheumatic Diseases.    2012:71(8):1322-1328.-   Laslett L L, Kingsbury S R. Hensor E M A, Bowes M A. Conaghan P G.    Effect of bisphosphonate use in patients with symptomatic and    radiographic knee osteoarthritis: data from the Osteoarthritis    Initiative. Annals of the Rheumatic Diseases. 2014; 73(5):824-830.-   Liu, C., Li, Q., Su, Y. & Bao, L. Prostaglandin E2 promotes Na1.8    trafficking via its intracellular RRR motif through the protein    kinase A pathway. Traffic (Copenhagen, Denmark) 11, 405-417 (2010).-   Lotz, J. C., Fields, A. J. & Liebenberg, E. C. The role of the    vertebral end plate in low back pain. Global Spine J 3, 153-164    (2013).-   Luoma, K., et al., Low back pain in relation to lumbar disc    degeneration. Spine (Phila Pa. 1976), 2000. 25(4): p. 487-92.-   Luoma, K., Vehmas, T., Kerttula, L., Gronblad, M. & Rinne, E.    Chronic low back pain in relation to Modic changes, bony endplate    lesions, and disc degeneration in a prospective MRI study. European    spine journal:official publication of the European Spine Society,    the European Spinal Deformity Society, and the European Section of    the Cervical Spine Research Society 25, 2873-2881 (2016).-   Lyu G, Guan Y. Zhang C, et al. TGF-beta signaling alters H4K20me3    status via miR-29 and contributes to cellular senescence and cardiac    aging. Nat Commun. 2018; 9(1):2560.-   Ma L, Wu J, Jin Q H. The association between parathyroid hormone    1-34 and the Wnt/β-catenin signaling pathway in a rat model of    osteoarthritis. Mol Med Rep. 2017:16(6):8799-8807.-   Maatta, J. H., Wadge, S., MacGregor, A., Karppinen, J. &    Williams, F. M. ISSLS Prize Winner: Vertebral Endplate (Modic)    Change is an Independent Risk Factor for Episodes of Severe and    Disabling Low Back Pain. Spine (Phila Pa. 1976) 40, 1187-1193,    doi:10.1097/BRS.0000000000000937 (2015).-   Maher, C., M. Underwood, and R. Buchbinder, Non-specific low back    pain. Lancet, 2017. 389(10070): p. 736-747.-   Malfait A M. Schnitzer T J. Towards a mechanism-based approach to    pain management in osteoarthritis. Nature Reviews Rheumatology.    2013; 9(11):654-664.-   Manchikanti, L., Epidemiology of low back pain. Pain    Physician, 2000. 3(2): p. 167-92.-   Mapp P I, Walsh D A. Mechanisms and targets of angiogenesis and    nerve growth in osteoarthritis. Nature Reviews Rheumatology. 2012;    8(7):390-398.-   Masuda, K. et al. A novel rabbit model of mild, reproducible disc    degeneration by an annulus needle puncture: Correlation between the    degree of disc injury and radiological and histological appearances    of disc degeneration. Spine 30, 5-14 (2005).-   McDougall J J, Andruski B. Schuelert N, Hallgrímsson B, Matyas J R    Unravelling the relationship between age, nociception and joint    destruction in naturally occurring osteoarthritis of Dunkin Hartley    guinea pigs. Pain. 2009; 141(3):222-232.-   Mease P J, Hanna S, Frakes E P, Altman R D. Pain Mechanisms in    Osteoarthritis: Understanding the Role of Central Pain and Current    Approaches to Its Treatment. The Journal of Rheumatology. 2011;    38(8):1546.-   Millecamps, M., Czerminski, J. T., Mathieu, A. P. & Stone, L. S.    Behavioral signs of axial low back pain and motor impairment    correlate with the severity of intervertebral disc degeneration in a    mouse model. Spine J 15, 2524-2537, doi:10.1016/j.spinee.2015.08.055    (2015).-   Miller R E, Ishihara S, Bhattacharyya B, et al. Chemogenetic    Inhibition of Pain Neurons in a Mouse Model of Osteoarthritis.    Arthritis & rheumatology (Hoboken, N.J.). 2017; 69(7):1429-1439.-   Miller, J. A., C. Schmatz, and A. B. Schultz, Lumbar disc    degeneration: correlation with age, sex, and spine level in 600    autopsy specimens. Spine (Phila Pa. 1976). 1988. 13(2): p. 173-8.-   Miyakoshi, N., et al., Evidence that anabolic effects of PTH on bone    require IGF-I in growing mice. Endocrinology, 2001. 142(10): p.    4349-56.-   Miyamoto, S., Yonenobu, K. & Ono, K. Experimental cervical    spondylosis in the mouse. Spine 16, S495-500 (1991).-   Moore, S. W., Zhang, X., Lynch, C. D. & Sheetz, M. P. Netrin-1    attracts axons through FAK-dependent mechanotransduction. The    Journal of neuroscience:the official journal of the Society for    Neuroscience 32, 11574-11585 (2012).-   Nakao, K. et al. CJ-023,423, a novel, potent and selective    prostaglandin EP4 receptor antagonist with antihyperalgesic    properties. The Journal of pharmacology and experimental    therapeutics 322, 686-694 (2007).-   Nakashima, T et al. Evidence for osteocyte regulation of bone    homeostasis through RANKL expression. Nat Med 17, 1231-1234 (2011).-   Ohtori, S., Inoue, G., Miyagi, M. & Takahashi, K. Pathomechanisms of    discogenic low back pain in humans and animal models. Spine J 15,    1347-1355 (2015).-   Orth P. Cucchiarini M. Wagenpfeil S. Menger M D. Madry H. PTH    [1-34]-induced alterations of the subchondral bone provoke early    osteoarthritis. Osteoarthritis and cartilage. 2014; 22(6):813-821.-   Orth P, Cucchiarini M, Zurakowski D. Menger M D, Kohn D M. Madry H.    Parathyroid hormone [1-34] improves articular cartilage surface    architecture and integration and subchondral bone reconstitution in    osteochondral defects in vivo. Osteoarthritis and cartilage. 2013;    21(4):614-624.-   Papadakis, M., Sapkas, G., Papadopoulos, E. C. & Katonis, P.    Pathophysiology and biomechanics of the aging spine. Open Orthop J    5, 335-342. doi:10.2174/1874325001105010335 (2011).-   Park, K. W. et al. The axonal attractant Netrin-1 is an angiogenic    factor. Proceedings of the National Academy of Sciences of the    United States of America 101, 16210-16215 (2004).-   Peat G, McCarney R, Croft P. Knee pain and osteoarthritis in older    adults: a review of community burden and current use of primary    health care. Annals of the Rheumatic Diseases. 2001; 60(2):91-97.-   Pfeilschifter J, Laukhuf F, Muller-Beckmann B, Blum W F, Pfister T,    Ziegler R. Parathyroid hormone increases the concentration of    insulin-like growth factor-I and transforming growth factor beta 1    in rat bone. The Journal of clinical investigation. 1995;    96(2):767-774.-   Philipot D, Guerit D. Platano D, et al. p161NK4a and its regulator    miR-24 link senescence and chondrocyte terminal    differentiation-associated matrix remodeling in osteoarthritis.    Arthritis research & therapy. 2014; 16(1):R58.-   Pinto, V., Szucs, P., Derkach, V. A. & Safronov, B V. Monosynaptic    convergence of C- and Adelta-afferent fibres from different    segmental dorsal roots on to single substantia gelatinosa neurones    in the rat spinal cord. J Physiol 586, 4165-4177, doi:    10.1113/jphysiol.2008.154898 (2008).-   Powell, M. C., et al., Prevalence of lumbar disc degeneration    observed by magnetic resonance in symptomless women. Lancet, 1986.    2(8520): p. 1366-7.-   Prevalence and most common causes of disability among adults-United    States, 2005. MMWR. Morbidity and mortality weekly report. 2009;    58(16):421-426.-   Prisby R, Guignandon A, Vanden-Bossche A, et al. Intermittent    PTH(1-84) is osteoanabolic but not osteoangiogenic and relocates    bone marrow blood vessels closer to bone-forming sites. Journal of    bone and mineral research:the official journal of the American    Society for Bone and Mineral Research. 2011:26(11):2583-2596.-   Pritzker K P, Gay S, Jimenez S A, et al. Osteoarthritis cartilage    histopathology: grading and staging. Osteoarthritis and cartilage.    2006; 14(1):13-29.-   Qiu T, Wu X, Zhang F, Clemens T L, Wan M, Cao X. TGF-beta type II    receptor phosphorylates PTH receptor to integrate bone remodelling    signalling. Nature cell biology. 2010; 12(3):224-234.-   Rahme, R. & Moussa, R. The Modic vertebral endplate and marrow    changes: Pathologic significance and relation to low back pain and    segmental instability of the lumbar spine. American Journal of    Neuroradiology 29, 838-842, doi:10.3174/ajnr.A0925 (2008).-   Rapisarda V. Borghesan M, Miguela V, et al. Integrin Beta 3    Regulates Cellular Senescence by Activating the TGF-beta Pathway.    Cell reports. 2017; 18(10):2480-2493.-   Reilly K A, Beard D J. Barker K L, Dodd C A F, Price A J, Murray    D W. Efficacy of an accelerated recovery protocol for Oxford    unicompartmental knee arthroplasty—a randomised controlled trial.    Knee. 2005; 12(5):351-357.-   Reimann 1, Christensen S B. A histological demonstration of nerves    in subchondral bone. Acta orthopaedica Scandinavica. 1977;    48(4):345-352.-   Rigaud, M. et al. Species and strain differences in rodent sciatic    nerve anatomy: implications for studies of neuropathic pain. Pain    136, 188-201, doi:10.1016/j.pain.2008.01.016 (2008).-   Roche B, Vanden-Bossche A, Malaval L, et al. Parathyroid hormone    1-84 targets bone vascular structure and perfusion in mice: impacts    of its administration regimen and of ovariectomy. Journal of bone    and mineral research:the official journal of the American Society    for Bone and Mineral Research. 2014; 29(7):1608-1618.-   Rodriguez, A. G., et al., Morphology of the human vertebral    endplate. J Orthop Res, 2012. 30(2): p. 280-7.-   Rubin, D. I. Epidemiology and risk factors for spine pain.    Neurologic Clinics 25, 353-+, doi:10.1016/j.ncl.2007.01.004 (2007).-   Saijilafu & Zhou, F.-Q. Genetic study of axon regeneration with    cultured adult dorsal root ganglion neurons. J Vis Exp (2012).-   Samartzis, D. & Grivas, T. B. Thematic series—Low back pain.    Scoliosis and Spinal Disorders 12, doi:ARTN    110.1186/s13013-016-0108-5 (2017).-   Sampson E R, Hilton M J, Tian Y, et al. Teriparatide as a    chondroregenerative therapy for injury-induced osteoarthritis.    Science translational medicine. 2011:3(101):101ra193.-   Schaible H G. Mechanisms of chronic pain in osteoarthritis. Current    rheumatology reports. 2012; 14(6):549-556.-   Schuelert, N. & McDougall, J. J. Involvement of Nav 1.8 sodium ion    channels in the transduction of mechanical pain in a rodent model of    osteoarthritis. Arthritis Res Ther 14, R5 (2012).-   Serafini, T. et al. Netrin-1 is required for commissural axon    guidance in the developing vertebrate nervous system. Cell 87.    1001-1014 (1996).-   Shu, T., Valentino, K. M., Seaman, C., Cooper, H. M. &    Richards, L. J. Expression of the netrin-1 receptor, deleted in    colorectal cancer (DCC), is largely confined to projecting neurons    in the developing forebrain. The Journal of comparative neurology    416, 201-212 (2000).-   Shuang, F. et al. Establishment of a rat model of lumbar facet joint    osteoarthritis using intraarticular injection of urinary plasminogen    activator. Sci Rep 5, 9828, doi:10.1038/srep09828 (2015).-   Soleimani M. Nadri S. A protocol for isolation and culture of    mesenchymal stem cells from mouse bone marrow. Nature protocols.    2009; 4(1):102-106.-   Southall, M. D. & Vasko, M. R. Prostaglandin receptor subtypes, EP3C    and EP4, mediate the prostaglandin E2-induced cAMP production and    sensitization of sensory neurons. J Biol Chem 276, 16083-16091    (2001).-   Suokas A K. Walsh D A, McWilliams D F, et al. Quantitative sensory    testing in painful osteoarthritis: a systematic review and    meta-analysis. Osteoarthritis and cartilage. 2012; 2010):1075-1085.-   Sun S. Gill S E, Massena de Camin S. Wilson D, McWilliams D F, Walsh    D A. Neurovascular invasion at the osteochondral junction and in    osteophytes in osteoarthritis. Annals of the rheumatic diseases.    2007; 66(11):1423-1428.-   Taher, F. et al. Lumbar degenerative disc disease: current and    future concepts of diagnosis and management. Adv Orthop 2012,    970752, doi:10.1155/2012/970752 (2012).-   Tang Y, Wu X, Lei W, et al. TGF-beta1-induced migration of bone    mesenchymal stem cells couples bone resorption with formation.    Nature medicine. 2009; 15(7):757-765.-   Telleria-Diaz A, Schmidt M, Kreusch S, et al. Spinal antinociceptive    effects of cyclooxygenase inhibition during inflammation:    Involvement of prostaglandins and endocannabinoids. Pain. 2010;    148(1):26-35.-   Thomas A, Eichenberger G. Kempton C, et al. Recommendations for the    treatment of knee osteoarthritis, using various therapy techniques,    based on categorizations of a literature review. Journal of    geriatric physical therapy (2001). 2009:32(1):33-38.-   Tomlinson R E, Li Z, Zhang Q, et al. NGF-TrkA Signaling by Sensory    Nerves Coordinates the Vascularization and Ossification of    Developing Endochondral Bone. Cell reports. 2016; 16(10):2723-2735.-   Traub, R. J. & Mendell, L. M. The spinal projection of individual    identified A-delta- and C-fibers. J Neurophysiol 59, 41-55,    doi:10.1152/jn.1988.59.1.41 (1988).-   Tu, T. et al. CD146 acts as a novel receptor for netrin-1 in    promoting angiogenesis and vascular development. Cell research 25,    275-287 (2015).-   Usoskin D. Furlan A. Islam S, et al. Unbiased classification of    sensory neuron types by large-scale single-cell RNA sequencing.    Nature neuroscience. 2015; 18(1):145-153.-   Vangsness C T, Jr., Spiker W. Erickson J. A review of evidence-based    medicine for glucosamine and chondroitin sulfate use in knee    osteoarthritis. Arthroscopy:the journal of arthroscopic & related    surgery:official publication of the Arthroscopy Association of North    America and the International Arthroscopy Association. 2009;    25(1):86-94.-   Videman, T., et al., The long-term effects of physical loading and    exercise lifestyles on back-related symptoms, disability, and spinal    pathology among men. Spine (Phila Pa. 1976), 1995. 20(6): p.    699-709.-   Wan, M., et al., Parathyroid hormone signaling through low-density    lipoprotein-related protein 6. Genes Dev, 2008. 22(21): p. 2968-79.-   Wang, Y., Videman, T. & Battie, M. C. ISSLS Prize Winner: Lumbar    Vertebral Endplate Lesions Associations With Disc Degeneration and    Back Pain History. Spine 37, 1490-1496,    doi:10.10971BRS.0b013e3182608ac4 (2012).-   Wei, F., et al., Pingyangmycin-induced in vivo lumbar disc    degeneration model of rhesus monkeys. Spine (Phila Pa. 1976), 2015.    40(4): p. E199-210.-   Wein, M. N. and H. M. Kronenberg, Regulation of Bone Remodeling by    Parathyroid Hormone. Cold Spring Harb Perspect Med, 2018. 8(8).-   Wenger, H. C. and A. S. Cifu, Treatment of Low Back Pain.    JAMA, 2017. 318(8): p. 743-744.-   Woolf C J, Salter M W. Neuronal Plasticity: Increasing the Gain in    Pain. 2000; 288(5472):1765-1768.-   Woolf C J. Central sensitization: Implications for the diagnosis and    treatment of pain. Pain. 2011:152(3, Supplement):S2-S15.-   Wrana J L, Attisano L. Carcamo J, et al. TGF beta signals through a    heteromeric protein kinase receptor complex. Cell. 1992;    71(6):1003-1014.-   Wright A A, Cook C, Abbott J H. Variables associated with the    progression of hip osteoarthritis: a systematic review. Arthritis    and rheumatism. 2009; 61(7):925-936.-   Xie H, Cui Z, Wang L. et al. PDGF-BB secreted by preosteoclasts    induces angiogenesis during coupling with osteogenesis. Nature    Medicine. 2014; 20(11):1270-1278.-   Xiong, J. et al. Matrix-embedded cells control osteoclast formation.    Nat Med 17, 1235-1241 (2011).-   Yan J Y, Tian F M, Wang W Y, et al. Parathyroid hormone (1-34)    prevents cartilage degradation and preserves subchondral bone    micro-architecture in guinea pigs with spontaneous osteoarthritis.    Osteoarthritis and cartilage. 2014:22(11):1869-1877.-   Yu B, Zhao X, Yang C, et al. Parathyroid hormone induces    differentiation of mesenchymal stromal/stem cells by enhancing bone    morphogenetic protein signaling. 2012; 27(9):2001-2014.-   Yusuf E, Kortekaas M C, Watt I, Huizinga T W J, Kloppenburg M. Do    knee abnormalities visualised on MRI explain knee pain in knee    osteoarthritis? A systematic review. Annals of the Rheumatic    Diseases. 2011:70(1).60-67.-   Zhang W, Moskowitz R W, Nuki G, et al. OARSI recommendations for the    management of hip and knee osteoarthritis. Part 11: OARSI    evidence-based, expert consensus guidelines. Osteoarthritis and    cartilage. 2008; 16(2):137-162.-   Zhen G, Wen C. Jia X, et al. Inhibition of TGF-beta signaling in    mesenchymal stem cells of subchondral bone attenuates    osteoarthritis. Nature medicine. 2013:19(6):704-712.-   Zhen G H, Cao X. Targeting TGF beta signaling in subchondral bone    and articular cartilage homeostasis. Trends in Pharmacological    Sciences. 2014:35(5):227-236.-   Zheng, L., et al., Ciliary parathyroid hormone signaling activates    transforming growth factor-beta to maintain intervertebral disc    homeostasis during aging. Bone Res, 2018. 6: p. 21.-   Zhong, R., et al., The effects of intervertebral disc degeneration    combined with osteoporosis on vascularization and microarchitecture    of the endplate in rhesus monkeys. Eur Spine J, 2016. 25(9): p.    2705-15.-   Zhou, M., et al., Mortality, morbidity, and risk factors in China    and its provinces, 1990-2017: a systematic analysis for the Global    Burden of Disease Study 2017. Lancet. 2019.-   Zhou, Z., et al, Intervertebral disk degeneration: T1rho M R imaging    of human and animal models. Radiology. 2013. 268(2): p. 492-500.-   Zhu S, Zhu J, Zhen G. et al. Subchondral bone osteoclasts induce    sensory innervation and osteoarthritis pain. The Journal of clinical    investigation. 2019:129(3):1076-1093.

Although the foregoing subject matter has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be understood by those skilled in the art thatcertain changes and modifications can be practiced within the scope ofthe appended claims.

That which is claimed:
 1. A method for treating low back pain (LBP)and/or osteoarthritic pain in a subject in need of treatment thereof,the method comprising administering to the subject a compositioncomprising a recombinant parathyroid hormone (PTH) and apharmaceutically acceptable carrier.
 2. The method of claim 1, whereinthe low back pain comprises a nonspecific low back pain.
 3. The methodof claim 1 or claim 2, wherein administering the composition comprisingthe recombinant parathyroid hormone (PTH) inhibits osteoclastactivity-induced sensory innervation in a vertebral endplate of thesubject.
 4. The method of claim 1 or claim 2, wherein administering thecomposition comprising the recombinant parathyroid hormone (PTH)increases the intervertebral disc (IVD) space by decreasing the volumeand porosity of sclerotic endplates.
 5. The method of claim 1 or claim2, wherein administering the composition comprising recombinantparathyroid hormone (PTH) prevents endplate remodeling and sclerosis. 6.The method of claim 1 or claim 2, wherein administering the compositioncomprising the recombinant parathyroid hormone (PTH) reduces sensorynerve fibers.
 7. The method of claim 1 or claim 2, wherein administeringthe composition comprising the recombinant parathyroid hormone (PTH)reduces the porosity of sclerotic endplates.
 8. The method of any one ofclaims 1-7, wherein administering the composition comprising therecombinant parathyroid hormone (PTH) treats the osteoarthritic pain byone or more of inhibition of nerve innervation, inhibition ofsubchondral bone deterioration, inhibition of articular cartilagedegeneration, attenuation of joint degeneration, deceleratingsubchondral bone deterioration, and sustaining of subchondral bonemicroarchitecture by remodeling.
 9. The method of any one of claims 1-8,further comprising administering at least one other agent in combinationwith administering the composition comprising the recombinantparathyroid hormone (PTH).
 10. The method of claim 9, wherein the atleast one other agent is selected from paracetamol, an opioid, anon-steroidal anti-inflammatory drug, a skeletal muscle relaxant, atriptan, an α2-agonist, a local anesthetic, a tricyclic antidepressant,a benzodiazepine, a steroid, a visco supplement, and combinationsthereof.
 11. The method of any one of claims 1-10, wherein the low backpain is associated with one or more of spine degeneration, lumbar discherniation (LDH), scoliosis, cancer, and an infection.
 12. The method ofany one of claims 1-11, wherein the recombinant PTH comprises afull-length PTH protein or a fragment of PTH.
 13. The method of claim 1,wherein the recombinant parathyroid hormone comprises teriparatide(PTH(34′)).
 14. The method of claim 1, wherein the recombinantparathyroid hormone comprises an intact parathyroid hormone (iPTH). 15.The method of any one of claims 1-14, wherein the composition isadministered to the subject at least once a day.
 16. Use of arecombinant parathyroid hormone to treat low back pain (LBP) orosteoarthritic pain in a subject in need thereof.